Antibodies to endoplasmin and their use

ABSTRACT

Isolated monoclonal antibodies are disclosed herein that specifically bind endoplasmin. In some embodiments these antibodies are fully human. Recombinant nucleic acids encoding these antibodies, expression vectors including these nucleic acids, and host cells transformed with these expression vectors are also disclosed herein. In several embodiments the disclosed antibodies are of use for detecting and/or treating tumors that express endoplasmin, such as melanoma, breast cancer, head and neck squamous cell carcinoma, renal cancer, lung cancer, glioma, bladder cancer, ovarian cancer or pancreatic cancer. In one example, the tumor is a melanoma.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.13/161,432, filed Jun. 15, 2011, which claims the benefit of U.S.Provisional Application No. 61/355,516, filed Jun. 16, 2010. The priorapplications are incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States government support pursuantto Grant No. CA105500 and Grant No. CA138188 from the NationalInstitutes of Health. The United States government has certain rights inthe invention.

FIELD

This relates to the field of antibodies, specifically to fully humanantibodies that specifically bind endoplasmin.

BACKGROUND

Melanomas are aggressive, frequently metastatic tumors derived fromeither melanocytes or melanocyte related nevus cells (“Cellular andMolecular Immunology” (1991) (eds.) Abbas A. K., Lechtman, A. H., Pober,J. S.; W. B. Saunders Company, Philadelphia: pages 340-341). Melanomasmake up approximately three percent of all skin cancers and theworldwide increase in melanoma is unsurpassed by any other neoplasm withthe exception of lung cancer in women (“Cellular and MolecularImmunology” (1991) (eds.) Abbas, A. K., Lechtiman, A. H., Pober, J. S.;W. B. Saunders Company Philadelphia pages: 340-342; Kirkwood andAgarwala (1993) Principles and Practice of Oncology 7:1-16). Even whenmelanoma is apparently localized to the skin, up to 30% of the patientswill develop systemic metastasis and the majority will die (Kirkwood andAgarwala (1993) Principles and Practice of Oncology 7:1-16). Classicmodalities of treating melanoma include surgery, radiation andchemotherapy. In the past decade, immunotherapy and other molecularmethods have emerged as new and promising methods for treating melanoma.

Strong evidence that an immune response to cancer exists in humans isprovided by the existence of lymphocytes within melanoma deposits. Theselymphocytes, when isolated, are capable of recognizing specific tumorantigens on autologous and allogeneic melanomas in a majorhistocompatibility complex (MHC)-restricted fashion (Itoh et al. (1986),Cancer Res. 46: 3011-3017; Muul et al. (1987), J. Immunol. 138:989-995);Topalian et al. (1989) J. Immunol. 142: 3714-3725; Darrow et al. (1989)J. Immunol. 142: 3329-3335; Hom et al. (1991) J. Immunother. 10:153-164;Kawakami et al. (1992) J. Immunol. 148: 638-643; Hom et al. (1993) J.Immunother. 13:18-30; O'Neil et al. (1993) J. Immunol. 151: 1410-1418).Tumor infiltrating lymphocytes (TIL) from patients with metastaticmelanoma recognize shared antigens including melanocyte-melanoma lineagespecific tissue antigens in vitro (Kawakami et al. (1993) J. Immunother.14: 88-93; Anichini et al. (1993) J. Exp. Med. 177: 989-998). The factthat many melanoma patients mount cellular and humoral responses againstthese tumors and that melanomas express both MHC antigens and tumorassociated antigens (TAA) suggests that identification andcharacterization of additional melanoma antigens will be important forimmunotherapy of patients with melanoma. However, there remains a needfor new modalities for the treatment of melanoma and other cancers.

SUMMARY

Isolated monoclonal antibodies and antigen binding fragments of theseantibodies are disclosed herein that specifically bind endoplasmin(Grp94). In some embodiments these antibodies are fully human. Theseantibodies have a high affinity for human endoplamin and can be used totreat and/or diagnose cancer. In one example, the monoclonal antibody isan scFv.

In some embodiments the disclosed antibodies are of use for detectingtumors that express endoplasmin, such as melanoma, breast cancer, headand neck squamous cell carcinoma, renal cancer, lung cancer, glioma,bladder cancer, ovarian cancer or pancreatic cancer. In otherembodiments, the disclosed antibodies are of use for treating a tumor,such as melanoma, breast cancer, head and neck squamous cell carcinoma,renal cancer, lung cancer, glioma, bladder cancer, ovarian cancer orpancreatic cancer.

Recombinant nucleic acids encoding these antibodies, expression vectorsincluding these nucleic acids, and host cells transformed with theseexpression vectors are also disclosed herein.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a. Panning of a phage display scFv library with the humanmelanoma cell line WM1158. The phage display scFv library contains alarge of phage displaying scFv fragments with different specificity. Thelibrary was added to a tube containing WM1158 melanoma cell suspension.Following washing of the tube to remove unbound phage, bound phage wereeluted at a high pH and amplified in bacterial host E. coli TG1.Following three rounds of panning, the isolated clones were absorbedwith cultured human LG2 B lymphoid cells to remove the phage that boundto the Ags shared by human melanoma and lymphoid cells. The isolatedphage were then screened for reactivity with WM1158 cells in ELISA.

FIG. 1 b. Differential reactivity with the melanoma cell line WM1158 andwith the B lymphoid cell line LG2 of the scFv W9 isolated by panning aphage display antibody library with WM1158 cells. WM1158 cells wereplated in a 96-well plate and incubated with scFv W9 for 2 hours at roomtemperature. The binding of scFv was detected using c-myc-specific mAb9E10 and HPR-streptavidin. scFv 119, which recognizes an irrelevantantigen and LG2 cells were used as negative controls. scFv W9 reactsspecifically with WM1158 cell line.

FIG. 2. Reactivity of scFv W9 with many types of human cell lines. Sixmelanoma, four breast, one head and neck squamous cell, three pancreas,one bladder, one lung, one epithelial, one colon, one renal, oneprostate, and one ovarian cancer cell lines were plated in a 96-wellplate and incubated with scFv W9 for 2 hours at room temperature. Thebinding of scFv was detected using c-myc-specific mAb 9E10 andHPR-streptavidin. scFv W9 reacted with all the melanoma cell lines, withtwo breast cancer cell lines, and with head and neck squamous cell,pancreas, bladder, lung, epithelial, renal, ovarian and glioma cancercell lines.

FIGS. 3 a and 3 b. Identification of endoplasmin as the antigenrecognized by scFv W9. A WM1158 melanoma cell lysate wasimmunoprecipitated with scFv W9. The -HLA class I-specific mAb TP25.99,and the HMW-MAA-specific scFv C21 were used as controls. Proteins in theprecipitates were resolved on a reducing 10% SDS-PAGE and stained withCoomassie blue. The 94-KDa was unique to the W9 precipitate (A). Sameresults were obtained from lysates of T24 (bladder cancer), SUM149(breast cancer), and SLR21 (renal cancer) cell lines. The specific bandswere excised from the SDS gel and analyzed by mass spectrometry. Thehuman protein identified in the 94-KDa bands is endoplasmin.

FIG. 4. Role of carbohydrates in the expression of the determinantrecognized by scFv W9. COL038 cells were cultured for 72 hours in thepresence of 0.5 μg/ml of tunicamycin. Cells incubated in medium withDMSO alone, and the mAb TP25.99 were used as controls. Cells were testedby ELISA for the binding of scFv W9. Cells were incubated with scFv W9for 2 hours at 4° C. The binding of scFv was detected using mAb 9E10 andHPR-goat anti-mouse IgG antibodies. Absorbance was read at 450 nmTunicamycin treatment induced a strong decrease of scFv W9 binding toCOL038 cells. Thus, carbohydrates play a role in the expression of thedeterminant recognized by scFv W9.

FIGS. 5 a and 5 b. Specificity of the reactivity of scFv W9 torecombinant canine Endoplasmin (Grp94). Recombinant canine endoplasmin(Grp94), which displays a 98.5% homology in the amino acid sequence withhuman enodplasmin (Grp94), was immobilized in a 96-well plate at 20μg/well and incubated with scFv W9 for 2 hours at room temperature. Thebinding of scFv was detected using mAb 9E10 and HPR-goat anti-mouse IgGantibodies. Absorbance was read at 450 nm. scFv 119 and BSA were used asnegative controls. scFv W9 recognizes a determinant of endoplasminexpressed on the cell membrane.

FIG. 6. Dose-dependent inhibition by recombinant canine enodplamsin(Grp94) of scFv W9 binding to COL038 cells. Two fold dilutions ofrecombinant canine endoplasmin (Grp94) were preincubated with scFv W9.The mixture was added to 96-well plate seeded with COL038 cells. Thebinding of scFv was detected using mAb 9E10 and HPR— goat anti-mouse IgGantibodies. Absorbance was read at 450 nm. B₂m was used as a control.Recombinant canine Grp94 specifically inhibits the binding of scFv W9 toCOL038 cells.

FIGS. 7 a and 7 b. Effect of transfection of 293 cells with endoplasmin(Grp94) cDNA on binding of scFv W9. 293 cells were transfected with 3 μgof Grp94 HSP90B1 cDNA clone by electroporation. Cells were incubatedwith scFv W9 and mAb 9E10, followed by incubation with FITC-goatanti-mouse IgG antibodies. Cells were analyzed by flow cytometry. ThepCMV6-XL4 vector was used as a control. Untrasfected cells were used asa control. Electroporation increases the binding of scFv W9. Theexpression of the antigen recognized by scFv is regulated by heat shock.

FIGS. 8 a and 8 b. Effect of transduction of FO-1 cells with endoplasmin(Grp94) shRNA on binding of scFv W9. FO-1 cells were transduced withendoplasmin (Grp94) shRNA and a control shRNA (ABCB5). Cells wereincubated with scFv W9 and mAb 9E10, followed by incubation withFITC-goat anti-mouse IgG antibodies. Cells were then analyzed by flowcytometry. endoplasmin (Grp94) shRNA inhibited the binding of scFv W9compared with the control shRNA.

FIG. 9. Role of carbohydrates in the expression of the epitoperecognized by mAb W9. Human pancreatic adenocarcinoma MIAPaCa-2 (5×10⁵)were incubated with or without 20 of α-2(3,6,8.9)-Neuraminidase in 50 μlRPM I1640 medium for 24 hours at 37° C. in a 5% CO₂ incubator. Thetreated cells were then stained with mAb W9 and analyzed by flowcytometry. Cells treated with mAb TP25.99 were used as a control.

FIG. 10. Expression of the extracellular Endoplasmin (Grp94) epitoperecognized by mAb W9 on human pancreatic adenocarcinoma MIAPaCa-2 cancerinitiating cells. Human pancreatic adenocarcinoma MIAPaCa-2 cells wereincubated with ALDEFLUOR to detect ALDH activity (TEST), and stainedwith mAb W9. Cells incubated with ALDEFLUOR+DAEB inhibitor and stainedwith mAb W9 were used as a reference (CONTROL). Human Ig (HIg) were usedas a control. The percentage of cancer initiating cells, identified asALDH^(bright) cells, is indicated.

FIG. 11. Immunohistochemical staining by mAb W9 of a surgically removedhuman pancreatic adenocarcinoma lesion. Frozen sections of a surgicallyremoved human pancreatic adenocarcinoma lesion and normal pancreastissue from the same patient were stained with mAb W9 (1 μg/ml). (X200).

FIG. 12. IHC staining analysis of endoplasmin (Grp94) expression onhuman basal breast cancer MDA-MB-231 and human melanoma xenograft MV3 byusing mAb W9. Formalin fixed and paraffin embedded human basal breastcancer MDA-MB-231 cells, human luminal breast cancer MCF-7 cells andhuman melanoma xenograft MV3 were stained with mAb W9 (5 μg/ml) (X200).Immunoistochemical staining with mAb W9 showed that MDA-MB-231 cells andMV3 xenograft were strongly stained by mAb W9 (5 μg/ml). No staining wasdetected in the MCF-7 cells.

FIG. 13. mAb W9 significantly inhibited the growth of tumor cellsexpressing endoplasmin (Grp94). Human cancer cells (1×10⁴/well) wereseeded in a 96-well plate (RPMI 1640 media plus 1% FCS) and treated withmAb W9 (5 μg/ml) for 72 hours. Human Ig (HIg) were used as a control.Cells were then tested by MTT assay. The results are expressed as % ofgrowth inhibition. * p value <0.05; **p value <0.01.

FIG. 14. Induction by mAb W9 antibody of apoptosis in cancer cells.Human MV3 (melanoma) and MIAPaCa-2 (pancreatic adenocarcinoma) cells(4×10⁵/ml) were starved for 24 hrs and 3 hrs respectively, thenincubated with mAb W9 (50 μg/ml), in RPMI 1640 medium. containing 1.5%FCS. After 6 hrs cells were investigated for the percentage of apoptoticcells by staining with Annexin V/PI. Cells were analyzed by flowcytometry. Human Ig (HIg) were used as a negative control.

FIG. 15. Induction by mAb W9 antibody of cleaved PARP in human melanomaM21 cells. Human melanoma M21 cells (4×10⁵/ml) were incubated with mAbW9 (5 μg/ml), in RPMI 1640 medium containing 1.5% FCS for 72 hrs. Celllysates were tested in a Western blot analysis for cleaved PARP. β-actinwas used as the loading control. mAb W9 strongly increased theexpression of cleaved PARP.

FIG. 16. Induction by mAb W9 antibody of cleaved caspase-3 in humanmelanoma MV3 cells. Human melanoma MV3 cells (4×10⁵/ml) were starved for24 hrs, then incubated with mAb W9 (50 μg/ml), in RPMI 1640 mediumcontaining 1.5% FCS. Cell lysates were tested in Western blot forcleaved caspase-3. β-actin was used as the loading control. The densityof resultant bands was determined with IMAGJ® software, normalized tothat of β-actin, are shown below the respective bands. mAb W9 stronglyincreased the expression of cleaved caspase-3. No effect was detected incells treated with HIg.

FIG. 17. Cell-dependent lysis of human melanoma MV3 cells mediated bymAb W9. Human melanoma MV3 cells were labeled with 50 μCi of ⁵¹Cr andresuspended at the density of 0.4×10⁶ cells/ml and combined with mAb W9(50, 10, 2 μg/ml) in a 96-well tissue culture-U-bottom assay plate.Human Ig (HIg) were used as a control. Following 30 minutes ofincubation at 4° C., PBMC (40:1 E:T) were added and incubated for 4 hrsat 37° C. in a CO₂ incubator. ⁵¹Cr release was determined by countingthe ell free supernatant in a Packard TOPCOUNT™ Microplate ScintillationCounter.

FIG. 18. Complement-dependent lysis of human melanoma MV3 cells mediatedby mAb W9. Human melanoma MV3 cells were labeled with 50 μCi of ⁵¹Cr andresuspended at the density of 1×10⁶ cells/ml. The target cells wereincubated with mAb W9 (50, 10, 2 μg/ml) in presence of human serumcomplement. Human Ig (Hig) were used as control. Following 2 hrs ofincubation at 37° C. in a CO₂ incubator, ⁵¹Cr release was determined bycounting the cell free supernatant in a Packard TOPCOUNT™ MicroplateScintillation Counter.

FIG. 19. Inhibition by mAb W9 of human pancreatic adenocarcinomaMIAPaCa-2 cancer initiating cell in vitro proliferation. Humanpancreatic adenocarcinoma MIAPaCa-2 cells were incubated with mAb W9 (25μg/ml), for 48 hrs at 37° C. Cells were then harvested and stained withALDEFLUOR® (TEST). Cells stained with ALDEFLUOR®+DAEB were used as areference (CONTROL). Human Ig (HIg) were used as a control. Thepercentage of cancer initiating cells, identified as ALDH^(bright)cells, is indicated.

FIG. 20. Inhibition by mAb W9 of signaling pathways RAS-MEK-ERK and FAKin pancreatic MIAPaCa-2 and PANC 1 cells. The human pancreatic MIAPaCa-2and PANC 1 cells were seeded at the concentration of 1.0×10⁵ per well ina 6-well plate in RPMI 1640 medium with 5% FCS and incubated with eitherthe W9 supernatant, the control supernatant, or untreated for 48 hrs at37° C. Cell lysate were tested in western blot with anti-RAS, C-Raf,phosphorylated (p)-ERK1/2, ERK1/2, (p)-FAK (Tyr397), FAK, β-catenin,p-AKT (Ser473) and AKT mAbs. Calnexin and β-actin was used as theloading control.

FIG. 21. Inhibition by mAb W9 of signaling pathways in human melanomaM21 cells. The human melanoma M21 cells were seeded at the concentrationof 1.0×10⁵ per well in a 6-well plate in RPMI 1640 medium with 5% FCSand incubated with mAb W9 (5 μg/ml) for 72 hrs. Cell lysate were testedin western blot with anti-phosphorylated (p)-AKT, Bc1-2, C-Raf,(p)-ERK1/2, PKCcc, β-catenin mAbs. Human Ig (HIg) and PBS were used asnegative controls. Calnexin was used as the loading control.

FIG. 22. Inhibition by mAb W9 of signaling pathways in human melanomaMV3 cells. Human melanoma MV3 cells were incubated with mAb W9 for 6 hrsat 37° C. in RPMI 1640. Cell lysate were then prepared and tested inwestern blot with anti-RAS, Met, p-Met, β-catenin, Ras, C-RAF, p-AKT,and p-ERK1/2. Thr202/Tyr204. β-actin was used as the loading control.Cells incubated with HIg were used as controls.

FIG. 23. Reduction of established lung metastasis in mice treated withmAb W9. MV3 melanoma cells (1.4×10⁸/mice) were injected i.v. After 15days mice were treated with mAb W9 (100 μg/mice, i.v.) every 48 hrs. Onday 25, mice were sacrificed, lungs were harvested, formalin fixed andH&E stained for the analysis of tumor areas. The values shown are themean tumor area of each group.** indicates p value <0.01.

FIG. 24. Enhancement by chemotherapeutic agents of endoplasmin (Grp94)expression by UACC-257 melanoma cells. Human melanoma UACC-257 cells(2×10⁵/ml) were incubated in RPMI 1640 medium containing 10% FCS with5-FU (300 μM), Cisplatin (10 μM), and Paclitaxel (20 nM) for 48 hrs.Cells were harvested, stained with mAb W9 and analyzed by flowcytometry. Untreated cells were used as control. Percentage of stainedcells and mean fluorescence intensity (MFI) are indicated.

FIGS. 25 a and 25 b. Inhibition by mAb W9 in combination with 5-FU andcyclopamine of human pancreatic adocarcinoma MIAPaCa-2 cellproliferation. Human pancreatic adenocarcinoma MIAPaCa-2 cells wereseeded (2.5×10³ cells per well) in a 96-well plate (RPMI 1640 media plus5% FCS) and treated with mAb W9 (5 μg/ml) in combination with 5-FU (10μM) (A.), or cyclopamine (20 μM) (B.) for 1, 2, 3 days at 37° C. in a 5%CO₂ atmosphere. Cells were then tested by MTT assay. The O.D. values at540 nm indicate the living cells.

FIG. 26. Inhibition by mAb W9 in combination with 5-FU and cyclopamineof human pancreatic adenocarcinoma MIAPaCa-2 cancer initiating cell invitro proliferation. Human pancreatic adenocarcinoma MIAPaCa-2 cellswere incubated with mAb W9 (25 μg/ml), cyclopamine (20 μM), and 5-FU (10μM) for 48 hrs at 37° C. Cells were then stained with ALDEFLUOR with orwithout the DEAB inhibitor to identify ALDH^(bright) cells. The anti-HLAclass I mAb TP25.99 was used as a control. The percentage of cancerinitiating cells, identified as ALDH^(bright) cells, is indicated.

FIG. 27. Induction by mAb W9 in combination with 5-FU and cyclopamine ofapoptosis in pancreatic adenocarcinoma MIAPaCa-2 cells. Human pancreaticadenocarcinoma MIAPaCa-2 cells (4×10⁵/ml) were starved for 3 hrs thenincubated with mAb W9 (10 μg/ml), cyclopamine (20 μM), and 5-FU (10 μM)in RPMI 1640 medium. containing 1.5% FCS. After 24 hrs cells wereinvestigated for the percentage of apoptotic cells by staining withAnnexin V/PI. Cells were analyzed by flow cytometry. HIg were used as anegative control.

FIGS. 28 a and 28 b. Inhibition by mAb W9 in combination with radiationand cyclopamine of human pancreatic adenocarcinoma MIAPaCa-2 cancerinitiating cell in vitro proliferation. Human pancreatic adenocarcinomaMIAPaCa-2 cells (4×10⁵/ml) were irradiated at the dose of 20Gy (panelA.) and incubated with mAb W9 (10 μg/ml) and cyclopamine (20 μM) for 72hrs at 37° C. Cells were then stained with ALDEFLUOR with or without theDEAB inhibitor to identify ALDH^(bright) cells. Non-irradiated cell wereused as a control (panel B.). The percentage of cancer initiating cells,identified as ALDH^(bright) cells, is indicated.

FIG. 29. Induction by mAb W9 in combination with radiation andcyclopamine of apoptosis in pancreatic adenocarcinoma MIAPaCa-2 cells.Human pancreatic adenocarcinoma MiaPaCa-2 cells (4×10⁵/ml) wereirradiated at the dose of 20Gy and incubated with mAb W9 (20 μg/ml) andcyclopamine (20 μM). After 8 hrs cells were tested for the percentage ofapoptotic cells by staining with Annexin V/PI. Cells were analyzed byflow cytometry. Non-irradiated cells and human Ig (HIg) were used ascontrols.

FIG. 30. Inhibition by mAb W9 in combination with 5-FU and cyclopamineof signaling pathways in human pancreatic adenocarcinoma MIAPaCa-2cells. Human pancreatic adenocarcinoma MIA PaCa-2 cells were incubatedwith the mAb W9, cyclopamine (20 μM) and 5-FU (10 μM) for 2 days at 37°C. (panel D). Cell lysate were then prepared and tested in western blotwith anti-RAS, C-Raf, phosphorylated (p)-MEK (Ser217/221), MEK,pERK(Thr202/Tyr204), ERK, p-AKT (Ser473), AKT mAbs. Calnexin was used asthe loading control. Cells incubated with mAb W9 alone (panel A), withmAb W9 and cyclopamine (panel B), and with mAb W9 and 5 FU (panel C)were used as controls.

FIG. 31. Inhibition by mAb W9 in combination with radiation andcyclopamine of signaling pathways in human pancreatic adenocarcinomaMIAPaCa-2 cells. Human pancreatic adenocarcinoma MIAPaCa-2 cells wereirradiated at the dose of 20Gy and incubated with mAb W9 (10 μg/ml) andcyclopamine (20 μM) for 48 hrs at 37° C. Cell lysates were then preparedand tested by western blot with anti-RAS, phosphorylated(p)-ERK(Thr202/Tyr204), ERK, p-AKT (Ser473), AKT, SHh, Gill mAbs.Calnexin was used as the loading control Calnexin and β-actin were usedas the loading control.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile [8123-85229-10_Sequence_Listing.txt, Jun. 26, 2013, 19.5 KB], whichis incorporated by reference herein.

SEQ ID NO: 1 is the amino acid sequence of the heavy chain of anantibody that specifically binds endoplasmin.

SEQ ID NO: 2 is the amino acid sequence of the light chain of anantibody that specifically binds endoplasmin.

SEQ ID NO: 3 is a nucleic acid sequence encoding the heavy chain of anantibody that specifically binds endoplasmin.

SEQ ID NO: 4 is a nucleic acid sequence of the light chain of anantibody that specifically binds endoplasmin.

SEQ ID NO: 5 is an amino acid sequence of a human endoplasmin.

SEQ ID NO: 6 is a nucleic acid sequence encoding human endoplasmin.

SEQ ID NOs: 7 and 8 are amino acid sequences of endoplasminpolypeptides.

DETAILED DESCRIPTION I. Abbreviations

5-FU: Fluorouracil

ADCC: antibody-dependent cell-mediated cytotoxicity

Ag: antigen

ALDH^(bright): Aldehyde Dehydrogenase (bright)

Annexin V: Annexin AS

β-catenin: cadherin-associated protein

B-Raf: Serine/threonine-protein kinase B-Raf

CDC: complement-directed cytotoxicity

CDR: complementarity determining region

C-Raf: RAF proto-oncogene serine/threonine-protein kinase

DEAB: 4-(diethylamino)benzaldehyde

DMEM: Dulbecco's modified Eagle's medium

ER: endoplasmic reticulum

ERK1/2: extracellular signal-regulated kinase1/2

FAK: focal adhesion kinase

FBS: fetal bovine serum

FR: framework region

GLI1: Glioma-associated oncogene homolog 1

Grp: Glucose-regulated protein

Gy: Gray

HRP: horse radish peroxidase

Ig: immunoglobulin

mAb: monoclonal antibody

MEK: Mitogen-activated protein kinase kinase

Met: c-Met

O.D.: optical density

PBS: phosphate buffered saline

p-ERK1/2: phosphorylated extracellular signal-regulated kinase1/2

p-FAK: phosphoryalted focal adhesion kinase

PI: Propidium iodide

RAS: RAt Sarcoma

scFv: single chain variable regions of both V_(H) and V_(L)

SHH: Sonic hedgehog homolog

V_(H): variable heavy chain region

V_(L): variable light chain region

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand specifically binds an epitope of an antigen, such as endoplasmin, ora fragment thereof. Antibodies are composed of a heavy and a lightchain, each of which has a variable region, termed the variable heavy(V_(H)) region and the variable light (V_(L)) region. Together, theV_(H) region and the V_(L) region are responsible for binding theantigen recognized by the antibody.

Antibodies include intact immunoglobulins and the variants. Functionalfragments (antigen-binding fragments) of antibodies, that specificallybind an antigen, such as endoplamin, are well known in the art, such asFab fragments, Fab′ fragments, F(ab)′₂ fragments, single chain Fvproteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”) thatspecifically bind the target antigen. A scFv protein is a fusion proteinin which a light chain variable region of an immunoglobulin and a heavychain variable region of an immunoglobulin are bound by a linker, whilein dsFvs, the chains have been mutated to introduce a disulfide bond tostabilize the association of the chains. The term also includesgenetically engineered forms such as chimeric antibodies (for example,humanized murine antibodies), heteroconjugate antibodies (such as,bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed.,W. H. Freeman & Co., New York, 1997. Functional fragments are alsotermed “antigen-binding” fragments, since they specifically bind thetarget antigen, such as human endoplasmin.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs has been defined (see, Kabat et al., Sequencesof Proteins of Immunological Interest, U.S. Department of Health andHuman Services, 1991, which is hereby incorporated by reference). TheKabat database is now maintained online and CDR sequences can bedetermined, for example, see IMGT/V-QUEST programme version: 3.2.18.,Mar. 29, 2011, available on the internet and Brochet, X. et al., Nucl.Acids Res. 36, W503-508, 2008). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies, such as humans. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three-dimensionalspace.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds endoplasmin generally willhave a specific V_(H) region and the V_(L) region sequence, and thusspecific CDR sequences. Antibodies with different specificities (i.e.different combining sites for different antigens) have different CDRs.Although it is the CDRs that vary from antibody to antibody, only alimited number of amino acid positions within the CDRs are directlyinvolved in antigen binding. These positions within the CDRs are calledspecificity determining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds endoplasmin.

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. A “humanized” immunoglobulin is animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (for example a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one embodiment, all the CDRs are from thedonor immunoglobulin in a humanized immunoglobulin. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. A “humanized antibody” is an antibody comprising a humanizedlight chain and a humanized heavy chain immunoglobulin. A humanizedantibody binds to the same antigen as the donor antibody that providesthe CDRs. The acceptor framework of a humanized immunoglobulin orantibody may have a limited number of substitutions by amino acids takenfrom the donor framework. Humanized or other monoclonal antibodies canhave additional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Humanized immunoglobulins can be constructed by means ofgenetic engineering (see for example, U.S. Pat. No. 5,585,089).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. An exemplary antigenis endoplasmin. The term “antigen” includes all related antigenicepitopes. “Epitope” or “antigenic determinant” refers to a site on anantigen to which B and/or T cells respond. Epitopes can be formed bothfrom contiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least three, andmore usually, at least five or eight to ten amino acids in a uniquespatial conformation. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue.Specific, non-limiting examples of a tissue specific antigen are amelanoma specific antigen, or a glioma, breast, lung, prostate, renal orbladder specific antigen. A disease-specific antigen is expressedcoincidentally with a disease process, such as melanoma or another typeof cancer. Specific non-limiting examples of a disease-specific antigenare an antigen whose expression correlates with, or is predictive of,tumor formation, such as melanoma and/or glioma, and/or another type ofcancer (for example, endoplasmin). A disease specific antigen may be anantigen recognized by T cells or B cells.

Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a specimen. An example of amplification is thepolymerase chain reaction, in which a biological sample collected from asubject is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification may be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals, including non-human primates. Similarly,the term “subject” includes both human and veterinary subjects.

Binding affinity: Affinity of an antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In another embodiment, a high binding affinity ismeasured by a competition radioimmunoassay. In another embodiment,binding affinity is measured by ELISA. An antibody that “specificallybinds” an antigen, such as endoplasmin with a high affinity and does notsignificantly bind other unrelated antigens.

Breast cancer: A neoplastic condition of breast tissue that can bebenign or malignant. The most common type of breast cancer is ductalcarcinoma. Ductal carcinoma in situ is a non-invasive neoplasticcondition of the ducts. Lobular carcinoma is not an invasive disease butis an indicator that a carcinoma may develop. Infiltrating (malignant)carcinoma of the breast can be divided into stages (I, IIA, IIB, IIIA,IIIB, and IV).

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In someembodiments, a chemotherapeutic agent is an agent of use in treatingbreast, melanoma, and/or gliomas. In one embodiment, a chemotherapeuticagent is radioactive compound. One of skill in the art can readilyidentify a chemotherapeutic agent of use (e.g. see Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 inAbeloff, Clinical Oncology 2^(nd) ed., © 2000 Churchill Livingstone,Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy,2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F,Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis,Mosby-Year Book, 1993). Combination chemotherapy is the administrationof more than one agent to treat cancer, such as the administration ofantibodies that specifically bind endoplasmin in combination with aradioactive or chemical compound to a subject.

Chimeric antibody: An antibody that includes sequences derived from twodifferent antibodies, which typically are of different species. Mosttypically, chimeric antibodies include human and murine antibodydomains, generally human constant regions and murine variable regions,murine CDRs and/or murine SDRs.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Chemotherapeutic agent: An agent with therapeutic usefulness in thetreatment of diseases characterized by abnormal cell growth (e.g., ananti-neoplastic agent). Such diseases include tumors, neoplasms, andcancer, as well as diseases characterized by hyperplastic growth such aspsoriasis. In one embodiment, a chemotherapeutic agent is an agent ofuse in treating neoplasms such as solid tumors. Chemotherapeutic agentscan be protein or non-protein agents, such as small molecule drugs,antibodies, peptides, proteins, and immunomodulators. In one embodiment,a chemotherapeutic agent is a radioactive molecule. One of skill in theart can readily identify a chemotherapeutic agent (for instance, seeSlapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison'sPrinciples of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993).

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease the affinityof an antibody to specifically bind endoplasmin. For example, a humanantibody that specifically binds endoplasmin can include at most about1, at most about 2, at most about 5, and most about 10, or at most about15 conservative substitutions and specifically bind the originalendoplasmin polypeptide. The term conservative variation also includesthe use of a substituted amino acid in place of an unsubstituted parentamino acid, provided that antibody specifically binds endoplasmin.Non-conservative substitutions are those that reduce an activity orbinding to endoplasmin.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Complementarity determining region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,H-CDR2, H-CDR3, respectively.

Contacting: Placement in direct physical association; includes both insolid and liquid form.

Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to thecells intended to be targeted, as opposed to the cells of the rest of anorganism. In one embodiment, in contrast, the term “toxicity” refers totoxicity of an immunotoxin to cells other than those that are the cellsintended to be targeted by the targeting moiety of the immunotoxin, andthe term “animal toxicity” refers to toxicity of the immunotoxin to ananimal by toxicity of the immunotoxin to cells other than those intendedto be targeted by the immunotoxin.

Degenerate variant: A polynucleotide encoding an endoplasmin polypeptidethat includes a sequence that is degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequences areincluded in this disclosure as long as the amino acid sequence of theendoplasmin polypeptide encoded by the nucleotide sequence is unchanged.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, melanoma, ovarian cancer, breastcancer or a glioma. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of true positives).The “specificity” of a diagnostic assay is one minus the false positiverate, where the false positive rate is defined as the proportion ofthose without the disease who test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis. “Prognostic” is the probability of development (e.g.,severity) of a pathologic condition, such as a cancer or metastasis.

Effector molecule: The portion of a chimeric molecule that is intendedto have a desired effect on a cell to which the chimeric molecule istargeted. Effector molecule is also known as an effector moiety (EM),therapeutic agent, or diagnostic agent, or similar terms.

Therapeutic agents include such compounds as nucleic acids, proteins,peptides, amino acids or derivatives, glycoproteins, radioisotopes,lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeuticand diagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides. Alternatively, the molecule linkedto a targeting moiety, such as an anti-endoplasmin antibody, may be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (such as anantisense nucleic acid), or another therapeutic moiety that can beshielded from direct exposure to the circulatory system. Means ofpreparing liposomes attached to antibodies are well known to those ofskill in the art (see, for example, U.S. Pat. No. 4,957,735; and Connoret al., Pharm. Ther. 28:341-365, 1985). Diagnostic agents or moietiesinclude radioisotopes and other detectable labels. Detectable labelsuseful for such purposes are also well known in the art, and includeradioactive isotopes such as ³⁵S, ¹¹C_(,) ¹³N, ¹⁵O, ¹⁸F, ¹⁹F, ^(99m)Tc,¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I, fluorophores,chemiluminescent agents, and enzymes.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least five or eight to ten amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). An epitope canbe glycosylated. Thus, an antibody can specifically bind a glycosylatedform (or an unglycosylated form) of a protein.

Endoplasmin: A protein also known as Glucose-regulated protein (Grp) 94(Grp94), which is the endoplasmic reticulum (ER)-resident member of theheat-shock-protein 90 (Hsp90) family. In vivo, hsp90 and endoplasmininteract with client proteins and function to protect them fromubiquitin-dependent proteasomal degradation. Although the endoplasminprotein is expressed constitutively in all cell types, its expression isup-regulated under various stress conditions including low glucoselevels, low extracellular pH, expression of mutated proteins, and viralinfections. Heat-shock proteins have a cytoprotective function andmodulate apoptosis directly or indirectly.

It has been shown that cell surface expression of endoplasmin isincreased in tumor cells, including hepatocellular carcinoma, colorectalcarcinoma and lung cancer cells, and that endoplasmin has ananti-apoptotic effect on some tumor cells. Moreover, increased levels ofendoplasmin were observed when a chronic hepatitis B virus (HBV)infection progressed to cirrhosis and hepatocellular carcinoma (HCC).Inhibitors of Hsp90 and endoplasmin (such as geldanamycin (GA) and itsless toxic derivative 17-AAG) have been investigated for efficacy incancer treatment.

Exemplary nucleic acids encoding endoplasmin (Grp94) include, but arenot limited to: GENBANK® Accession Nos. NM_(—)003299, BC066656 (Homosapiens); NM_(—)011631 (Mus musculus); NM_(—)001045763: (Xenopus(Silurana) tropicalis); NM_(—)214103 (Sus scrofa) NM_(—)98210 (Daniorerio); NM_(—)001012197 (Rattus norvegicus); NM_(—)001134101: Pongoabelii; NM_(—)001003327 (Canis lupus familiaris) heat shock protein 90kDa beta (Grp94); NM_(—)204289 (Gallus gallus).

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Expressed: Translation of a nucleic acid into a protein. Proteins may beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Framework region: Amino acid sequences interposed between CDRs.Framework regions include variable light and variable heavy frameworkregions. The framework regions serve to hold the CDRs in an appropriateorientation for antigen binding.

Glioma: A tumor composed of neuroglia in any developmental state.Gliomas include all intrinsic neoplasms of the brain and spinal cord,such as astrocytomas, ependymomas, and oligodendrogliomas. “Low-grade”gliomas are well-differentiated (not anaplastic); these are benign andportend a better prognosis for the patient. “High-grade” gliomas areundifferentiated or anaplastic; these are malignant and carry a worseprognosis.

Glycosylation: The covalent attachment of a carbohydrate to a protein,such as an antigen. Glycosylation includes N-linked glycosylation,O-linked glycosylation and C-linked glycosylation.

HAMA (human anti-murine antibody) response: An immune response in ahuman subject to the variable and constant regions of a murine antibodythat has been administered to the patient. Repeated antibodyadministration may lead to an increased rate of clearance of theantibody from the patient's serum and may also elicit allergic reactionsin the patient.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody or functional fragment thereof that specifically binds anantigen of interest, such as human endoplasm. The effector molecule canbe a detectable label, an immunotoxin, a cytokine or a chemokine.Specific, non-limiting examples of toxins include, but are not limitedto, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38,and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxinsthereof, or other toxic agents that directly or indirectly inhibit cellgrowth or kill cells. For example, PE and DT are highly toxic compoundsthat typically bring about death through liver toxicity. PE and DT,however, can be modified into a form for use as an immunotoxin byremoving the native targeting component of the toxin (such as the domainIa of PE and the B chain of DT) and replacing it with a differenttargeting moiety, such as an antibody. A “chimeric molecule” is atargeting moiety, such as a ligand or an antibody, conjugated (coupled)to an effector molecule. The term “conjugated” or “linked” refers tomaking two polypeptides into one contiguous polypeptide molecule. In oneembodiment, an antibody is joined to an effector molecule. In anotherembodiment, an antibody joined to an effector molecule is further joinedto a lipid or other molecule to a protein or peptide to increase itshalf-life in the body. The linkage can be either by chemical orrecombinant means. In one embodiment, the linkage is chemical, wherein areaction between the antibody moiety and the effector molecule hasproduced a covalent bond formed between the two molecules to form onemolecule. A peptide linker (short peptide sequence) can optionally beincluded between the antibody and the effector molecule. Becauseimmunoconjugates were originally prepared from two molecules withseparate functionalities, such as an antibody and an effector molecule,they are also sometimes referred to as “chimeric molecules.” The term“chimeric molecule,” as used herein, therefore refers to a targetingmoiety, such as a ligand or an antibody, conjugated (coupled) to aneffector molecule.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence, such as an N-terminal repeat, such that the peptidewill bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”)response, or a B cell response (e.g. antibody production) against theantigen from which the immunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide. In one specific non-limitingexample, an immunogenic polypeptide includes a region of endoplasmin, ora fragment thereof, wherein the polypeptide that is expressed on thecell surface of a host cell that expresses the full-length endoplasminpolypeptide.

Immunogenic composition: A composition comprising a polypeptide, such asan endoplasmin polypeptide, that induces a measurable CTL responseagainst cells expressing endoplasmin polypeptide, or induces ameasurable B cell response (such as production of antibodies) against anendoplasmin polypeptide. An immunogenic composition can also inducecytokine production. It further refers to isolated nucleic acidsencoding an endoplasmin polypeptide that can be used to express theendoplasmin polypeptide (and thus be used to elicit an immune responseagainst this polypeptide). For in vitro use, an immunogenic compositionmay consist of the isolated protein or peptide epitope. For in vivo use,the immunogenic composition will typically comprise the protein orimmunogenic peptide in pharmaceutically acceptable carriers, and/orother agents. Any particular peptide, such as an endoplasminpolypeptide, or nucleic acid encoding the polypeptide, can be readilytested for its ability to induce a CTL or B cell response byart-recognized assays. Immunogenic compositions can include adjuvants,which are well known to one of skill in the art.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, and pH) that are typicalinside a living mammal or a mammalian cell. While it is recognized thatsome organs are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (i.e., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or organelle, has been substantiallyseparated or purified away from other biological components in theenvironment (such as a cell) in which the component naturally occurs,i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins andorganelles. Nucleic acids and proteins that have been “isolated” includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In one example, a “labeled antibody” refers to incorporationof another molecule in the antibody. For example, the label is adetectable marker, such as the incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionucleotides (such as ³⁵S, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F,^(99m)Tc, ¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I), fluorescentlabels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanidephosphors), enzymatic labels (such as horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (such as a leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags), or magnetic agents, such as gadolinium chelates. In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

Linker: In some cases, a linker is a peptide within an antibody bindingfragment (such as an Fv fragment) which serves to indirectly bond thevariable heavy chain to the variable light chain. “Linker” can alsorefer to a peptide serving to link a targeting moiety, such as anantibody, to an effector molecule, such as a cytotoxin or a detectablelabel.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an scFv. In the specific context, the terms include reference tojoining a ligand, such as an antibody moiety, to an effector molecule.The linkage can be either by chemical or recombinant means. “Chemicalmeans” refers to a reaction between the antibody moiety and the effectormolecule such that there is a covalent bond formed between the twomolecules to form one molecule.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Major histocompatibility complex (MHC): Generic designation meant toencompass the histocompatibility antigen systems described in differentspecies, including the human leukocyte antigens (“HLA”). The term“motif” refers to the pattern of residues in a peptide of definedlength, usually about 8 to about 11 amino acids, which is recognized bya particular MHC allele. The peptide motifs are typically different foreach MHC allele and differ in the pattern of the highly conservedresidues and negative binding residues.

Melanoma: A form of cancer that originates in melanocytes (cells thatmake the pigment melanin). Melanocytes are found primary in the skin,but are also present in the bowel and eye. Melanoma in the skin includessuperficial spreading melanoma, nodular melanoma, acral lentiginousmelanoma, and lentigo maligna (melanoma). Any of the above types mayproduce melanin or can be amelanotic. Similarly, any subtype may showdesmoplasia (dense fibrous reaction with neurotropism) which is a markerof aggressive behavior and a tendency to local recurrence. Othermelanomas include clear cell sarcoma, mucosal melanoma and uvealmelanoma.

Features that affect prognosis are tumor thickness in millimeters(Breslow's depth), depth related to skin structures (Clark level), typeof melanoma, presence of ulceration, presence of lymphatic/perineuralinvasion, presence of tumor infiltrating lymphocytes (if present,prognosis is better), location of lesion, presence of satellite lesions,and presence of regional or distant metastasis. When melanomas havespread to the lymph nodes, one of the most important factors is thenumber of nodes with malignancy. The extent of malignancy within a nodeis also important; micrometastases in which malignancy is onlymicroscopic have a more favorable prognosis than macrometastases. Whenthere is distant metastasis, the five year survival rate is less than 10percent; the median survival is 6 to 12 months.

Metastases to skin and lungs have a better prognosis. Metastases tobrain, bone and liver are associated with a worse prognosis.

Melanoma can be staged as follows:

-   -   Stage 0: Melanoma in Situ (Clark Level I), 100% Survival    -   Stage I/II: Invasive Melanoma, 85-95% Survival        -   T1a: Less than 1.00 mm primary, w/o Ulceration, Clark Level            II-III        -   T1b: Less than 1.00 mm primary, w/Ulceration or Clark Level            IV-V        -   T2a: 1.00-2.00 mm primary, w/o Ulceration    -   Stage II: High Risk Melanoma, 40-85% Survival        -   T2b: 1.00-2.00 mm primary, w/ Ulceration        -   T3a: 2.00-4.00 mm primary, w/o Ulceration        -   T3b: 2.00-4.00 mm primary, w/ Ulceration        -   T4a: 4.00 mm or greater primary w/o Ulceration        -   T4b: 4.00 mm or greater primary w/ Ulceration    -   Stage III: Regional Metastasis, 25-60% Survival        -   N1: Single Positive Lymph Node        -   N2: 2-3 Positive Lymph Nodes OR Regional Skin/In-Transit            Metastasis        -   N3: 4 Positive Lymph Nodes OR Lymph Node and Regional            Skin/In Transit Metastases    -   Stage IV: Distant Metastasis, 9-15% Survival        -   M1a:Distant Skin Metastasis, Normal lactate dehydrogenase            (LDH)        -   M1b: Lung Metastasis, Normal LDH        -   M1c: Other Distant Metastasis OR Any Distant Metastasis with            Elevated LDH

Monoclonal antibody: An antibody produced by a single clone ofB-lymphocytes, by a cell into which the light and heavy chain genes of asingle antibody have been transfected, or by a specific phage in anantibody library such that the monoclonal antibody includes a definedset of CDRs and specifically binds a target antigen of interest.Monoclonal antibodies are produced by methods known to those of skill inthe art, for instance (but not limited to) by making hybridantibody-forming cells from a fusion of myeloma cells with immune spleencells or selection from a phage display library of antibody sequences.Monoclonal antibodies include humanized and fully human monoclonalantibodies. As used herein, a functional fragment of a monoclonalantibody includes antibody fragments that specifically bind the targetprotein (antigen-binding) for the monoclonal antibody, such as, but notlimited to scFv, Fv, dsRv, or Fab. Monoclonal antibodies specificallybind an antigenic epitope, such as a glycosylated epitope. Monoclonalantibodies include bi-functional antibodies wherein one or more sets ofCDRs specifically binds a target antigen, such as endoplasmin, andeffector function enhanced Fc and other glycol-engineered antibodies.

Neoplasia, malignancy, cancer or tumor: The result of abnormal anduncontrolled growth of cells. Neoplasia, malignancy, cancer and tumorare often used interchangeably. The amount of a tumor in an individualis the “tumor burden” which can be measured as the number, volume, orweight of the tumor. A tumor that does not metastasize is referred to as“benign.” A tumor that invades the surrounding tissue and/or canmetastasize is referred to as “malignant.” Examples of hematologicaltumors include leukemias, including acute leukemias (such as11q23-positive acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myelogenous leukemia and myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronicleukemias (such as chronic myelocytic (granulocytic) leukemia, chronicmyelogenous leukemia, and chronic lymphocytic leukemia), polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent andhigh grade forms), multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer (including basal breast carcinoma,ductal carcinoma and lobular breast carcinoma), lung cancers, ovariancancer, prostate cancer, hepatocellular carcinoma, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,medullary thyroid carcinoma, papillary thyroid carcinoma,pheochromocytomas sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladdercarcinoma, and CNS tumors (such as a glioma, astrocytoma,medulloblastoma, craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma and retinoblastoma).

In several examples, a tumor is melanoma, breast cancer, renal cancer,glioma or a squamous cell carcinoma, such as head and neck cancer.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, such as a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (such as a promoter, origin ofreplication, ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, for example, by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, bythe homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see for example, Current Protocols in Molecular Biology(Ausubel et al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, such as version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The BLASTN program (for nucleotidesequences) uses as defaults a word length (W) of 11, alignments (B) of50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.The BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989).

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as a heterologous promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (such as a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (such as glycosylation orphosphorylation). In one embodiment, the polypeptide is endoplasminpolypeptide. A “residue” refers to an amino acid or amino acid mimeticincorporated in a polypeptide by an amide bond or amide bond mimetic. Apolypeptide has an amino terminal (N-terminal) end and a carboxyterminal (C-terminal) end.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop,such as a reduction in tumor burden or a deacrease in the number of sizeof metastases. “Ameliorating” refers to the reduction in the number orseverity of signs or symptoms of a disease, such as cancer.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, 15 nucleotides or more inlength. Primers may be annealed to a complementary target DNA strand bynucleic acid hybridization to form a hybrid between the primer and thetarget DNA strand, and then extended along the target DNA strand by aDNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother nucleic acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancers or repressor elements whichcan be located as much as several thousand base pairs from the startsite of transcription. Both constitutive and inducible promoters areincluded (see e.g., Bitter et al., Methods in Enzymology 153:516-544,1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the retrovirus long terminal repeat; theadenovirus late promoter; the vaccinia virus 7.5K promoter) may be used.Promoters produced by recombinant DNA or synthetic techniques may alsobe used. A polynucleotide can be inserted into an expression vector thatcontains a promoter sequence which facilitates the efficienttranscription of the inserted genetic sequence of the host. Theexpression vector typically contains an origin of replication, apromoter, as well as specific nucleic acid sequences that allowphenotypic selection of the transformed cells

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified nucleicacid is one in which the nucleic acid is more enriched than the nucleicacid in its natural environment within a cell. Similarly, a purifiedpeptide preparation is one in which the peptide or protein is moreenriched than the peptide or protein is in its natural environmentwithin a cell. Substantial purification denotes purification from otherproteins or cellular components. In one embodiment, a preparation ispurified (or isolated) such that the protein or peptide represents atleast 50% (such as, but not limited to, 70%, 80%, 90%, 95%, 98% or 99%)of the total peptide or protein content of the preparation. Theendoplasmin polypeptides disclosed herein can be purified (and/orsynthesized) by any of the means known in the art (see, e.g., Guide toProtein Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press,San Diego, 1990; and Scopes, Protein Purification: Principles andPractice, Springer Verlag, New York, 1982).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Recombinant toxins: Chimeric proteins in which a cell targeting moietyis fused to a toxin (Pastan et al., Science, 254:1173-1177, 1991). Ifthe cell targeting moiety is the Fv portion of an antibody, the moleculeis termed a recombinant immunotoxin (Chaudhary et al., Nature,339:394-397, 1989). The toxin moiety is genetically altered so that itcannot bind to the toxin receptor present on most normal cells.Recombinant immunotoxins selectively kill cells which are recognized bythe antigen binding domain. These recombinant toxins and immunotoxinscan be used to treat cancer, for example, a cancer in which endoplasminis expressed.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency, will vary depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific, non-limiting example of progressively higher stringencyconditions is as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). One of skill in the art can readily determine variations onthese conditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologues or variants of an endoplasmin polypeptide will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp,CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881,1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.Altschul et al., Nature Genet. 6:119, 1994, presents a detailedconsideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of an endoplasmin polypeptide are typicallycharacterized by possession of at least 75%, for example at least 80%,sequence identity counted over the full length alignment with the aminoacid sequence of endoplasmin using the NCBI Blast 2.0, gapped blastp setto default parameters. For comparisons of amino acid sequences ofgreater than about 30 amino acids, the Blast 2 sequences function isemployed using the default BLOSUM62 matrix set to default parameters(gap existence cost of 11, and a per residue gap cost of 1). Whenaligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95%, depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a endoplasmin specific binding agent is an agentthat binds substantially to an endoplasmin polypeptide. In oneembodiment, the specific binding agent is a monoclonal or polyclonalantibody that specifically binds endoplasmin.

Squamous cell carcinoma: A type of cancer that originates in squamouscells, thin, flat cells that form the surface of the skin, eyes, variousinternal organs, and the lining of hollow organs and ducts of someglands. Squamous cell carcinoma is also referred to as epidermoidcarcinoma. One type of squamous cell carcinoma is head and neck headsquamous cell carcinoma (HNSCC). Head and neck squamous cell carcinomaincludes cancers of the nasal cavity, sinuses, lips, mouth, salivaryglands, throat and larynx.

HNSCC can be staged as follows:

-   -   Stage 0: No evidence of tumor.    -   Stage I: Tumor is 2 cm or less in greatest dimension; no        evidence of regional lymph node involvement or distant        metastasis.    -   Stage II: Tumor is more than 2 cm, but no larger than 4 cm; no        evidence of regional lymph node involvement or distant        metastasis.    -   Stage III: Tumor is larger than 4 cm; in some cases, the tumor        has spread to the lymph nodes; no evidence of distant        metastasis.    -   Stage IV: Tumor has spread to the lymph nodes; in some cases,        distant metastases are present.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, often called“helper” T cells, help orchestrate the immune response, includingantibody responses as well as killer T cell responses. CD8⁺ T cellscarry the “cluster of differentiation 8” (CD8) marker. In oneembodiment, a CD8 T cell is a cytotoxic T lymphocytes. In anotherembodiment, a CD8 cell is a suppressor T cell.

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthof a tumor. In one embodiment, a therapeutically effective amount is theamount necessary to eliminate a tumor or decrease the number or size ofmetastases. When administered to a subject, a dosage will generally beused that will achieve target tissue concentrations (for example, intumors) that has been shown to achieve a desired in vitro effect.

Toxin: A molecule that is cytotoxic for a cell. Toxins include abrin,ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinumtoxin, saporin, restrictocin or gelonin, or modified toxins thereof. Forexample, PE and DT are highly toxic compounds that typically bring aboutdeath through liver toxicity. PE and DT, however, can be modified into aform for use as an immunotoxin by removing the native targetingcomponent of the toxin (such as domain Ia of PE or the B chain of DT)and replacing it with a different targeting moiety, such as an antibody.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Human Monoclonal Antibodies that Specifically Bind Endoplasmin

Antibodies have been produced that specifically bind endoplasmin(Grp94), including monoclonal antibodies, such as fully human monoclonalantibodies. These antibodies and/or antigen-biding fragments thereof canbe used to isolate endoplasmin, and can be used to detect and/or treattumors that express endoplasmin, such as, but not limited to, melanoma,breast cancer, head and neck squamous cell carcinoma, renal cancer, lungcancer, glioma, bladder cancer or pancreatic cancer. These antibodiescan be conjugated to detectable labels or effector molecules.

In one embodiment, the antibodies specifically bind glycosylatedendoplasmin. Thus, in this embodiment, the antibodies do notspecifically bind unglycosylated endoplasmin or unrelated antigens.

Disclosed herein are human monoclonal antibodies and antigen-bidingfragments thereof that specifically bind endoplasmin. In one example,human endoplasmin has an amino acid sequence set forth as:

SEQ ID NO: 5 MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIVVQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETAEDTTEDTEQDEDEEMD VGTDEEEETAKESTAEKDEL,See also GENBANK ® Accession No. NM_003299, asavailable on Jun. 16, 2010 incorporated herein by reference).

In another example, the Endoplasmin is encoded by the nucleic acidsequence set forth as:

SEQ ID NO: 6gtgggcggac cgcgcggctg gaggtgtgag gatccgaacc caggggtggg gggtggaggcggctcctgcg atcgaagggg acttgagact caccggccgc acgccatgag ggccctgtgggtgctgggcc tctgctgcgt cctgctgacc ttcgggtcgg tcagagctga cgatgaagttgatgtggatg gtacagtaga agaggatctg ggtaaaagta gagaaggatc aaggacggatgatgaagtag tacagagaga ggaagaagct attcagttgg atggattaaa tgcatcacaaataagagaac ttagagagaa gtcggaaaag tttgccttcc aagccgaagt taacagaatgatgaaactta tcatcaattc attgtataaa aataaagaga ttttcctgag agaactgatttcaaatgctt ctgatgcttt agataagata aggctaatat cactgactga tgaaaatgctctttctggaa atgaggaact aacagtcaaa attaagtgtg ataaggagaa gaacctgctgcatgtcacag acaccggtgt aggaatgacc agagaagagt tggttaaaaa ccttggtaccatagccaaat ctgggacaag cgagttttta aacaaaatga ctgaagcaca ggaagatggccagtcaactt ctgaattgat tggccagttt ggtgtcggtt tctattccgc cttccttgtagcagataagg ttattgtcac ttcaaaacac aacaacgata cccagcacat ctgggagtctgactccaatg aattttctgt aattgctgac ccaagaggaa acactctagg acggggaacgacaattaccc ttgtcttaaa agaagaagca tctgattacc ttgaattgga tacaattaaaaatctcgtca aaaaatattc acagttcata aactttccta tttatgtatg gagcagcaagactgaaactg ttgaggagcc catggaggaa gaagaagcag ccaaagaaga gaaagaagaatctgatgatg aagctgcagt agaggaagaa gaagaagaaa agaaaccaaa gactaaaaaagttgaaaaaa ctgtctggga ctgggaactt atgaatgata tcaaaccaat atggcagagaccatcaaaag aagtagaaga agatgaatac aaagctttct acaaatcatt ttcaaaggaaagtgatgacc ccatggctta tattcacttt actgctgaag gggaagttac cttcaaatcaattttatttg tacccacatc tgctccacgt ggtctgtttg acgaatatgg atctaaaaagagcgattaca ttaagctcta tgtgcgccgt gtattcatca cagacgactt ccatgatatgatgcctaaat acctcaattt tgtcaagggt gtggtggact cagatgatct ccccttgaatgtttcccgcg agactcttca gcaacataaa ctgcttaagg tgattaggaa gaagcttgttcgtaaaacgc tggacatgat caagaagatt gctgatgata aatacaatga tactttttggaaagaatttg gtaccaacat caagcttggt gtgattgaag accactcgaa tcgaacacgtcttgctaaac ttcttaggtt ccagtcttct catcatccaa ctgacattac tagcctagaccagtatgtgg aaagaatgaa ggaaaaacaa gacaaaatct acttcatggc tgggtccagcagaaaagagg ctgaatcttc tccatttgtt gagcgacttc tgaaaaaggg ctatgaagttatttacctca cagaacctgt ggatgaatac tgtattcagg cccttcccga atttgatgggaagaggttcc agaatgttgc caaggaagga gtgaagttcg atgaaagtga gaaaactaaggagagtcgtg aagcagttga gaaagaattt gagcctctgc tgaattggat gaaagataaagcccttaagg acaagattga aaaggctgtg gtgtctcagc gcctgacaga atctccgtgtgctttggtgg ccagccagta cggatggtct ggcaacatgg agagaatcat gaaagcacaagcgtaccaaa cgggcaagga catctctaca aattactatg cgagtcagaa gaaaacatttgaaattaatc ccagacaccc gctgatcaga gacatgcttc gacgaattaa ggaagatgaagatgataaaa cagttttgga tcttgctgtg gttttgtttg aaacagcaac gcttcggtcagggtatcttt taccagacac taaagcatat ggagatagaa tagaaagaat gcttcgcctcagtttgaaca ttgaccctga tgcaaaggtg gaagaagagc ccgaagaaga acctgaagagacagcagaag acacaacaga agacacagag caagacgaag atgaagaaat ggatgtgggaacagatgaag aagaagaaac agcaaaggaa tctacagctg aaaaagatga attgtaaattatactctcac catttggatc ctgtgtggag agggaatgtg aaatttacat catttctttttgggagagac ttgttttgga tgccccctaa tccccttctc ccctgcactg taaaatgtgggattatgggt cacaggaaaa agtgggtttt ttagttgaat tttttttaac attcctcatgaatgtaaatt tgtactattt aactgactat tcttgatgta aaatcttgtc atgtgtataaaaataaaaaa gatcccaaat, see also GENBANK ®Accession No. NM_003299, Jun. 16, 2010,incorporated herein by reference herein.

Once of skill in the art can readily use a nucleic acid sequence toproduce a polypeptide, such as endoplasmin using standard method inmolecular biology (see, for example, Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). With the therapeuticagents and antibodies described herein, one of skill in the art canreadily construct a variety of clones containing functionally equivalentnucleic acids, such as nucleic acids which differ in sequence but whichencode the same EM or antibody sequence. Thus, the present inventionprovides nucleic acids encoding antibodies and conjugates and fusionproteins thereof.

Described herein are isolated human monoclonal antibodies and fragmentsthereof that specifically bind human endoplasmin, such as glycosylatedendoplasmin. In some embodiments, the human monoclonal antibodyantigen-binding fragment is a scFv. Also described are compositionsincluding the provided human monoclonal antibodies or functionalfragment thereof (that specifically bind human endoplasmin) and apharmaceutically acceptable carrier. Nucleic acids encoding theseantibodies, expression vectors comprising these nucleic acids, andisolated host cells that express the nucleic acids are also provided.

Also described herein are immunoconjugates comprising the humanmonoclonal antibodies or antigen-binding fragment thereof thatspecifically binds human endoplasmin. The immunoconjugates can compriseany therapeutic agent, toxin or other moiety. In one example, the toxinis PE or a variant or fragment thereof. Compositions comprising theimmunoconjugates are also described.

Compositions comprising the human monoclonal antibodies thatspecifically bind endoplasmin or antigen-binding fragment thereof can beused for screening, research, detection and therapeutic purposes. Forexample, the human monoclonal antibodies or antigen-binding fragmentthereof can be used to identify other antibodies that specifically bindendoplasmin, such as in competitive immunoassays.

Compositions comprising the human monoclonal antibodies thatspecifically bind endoplasmin or an antigen-binding fragment thereof canbe used to treat a subject diagnosed with cancer, such as a cancer thatexhibits increased expression of endoplasmin relative to normal cells.For example, the antibodies can be used to treat melanoma, breastcancer, head and neck squamous cell carcinoma, renal cancer, lungcancer, glioma, bladder cancer, ovarian cancer or pancreatic cancer.Melanoma includes spreading melanoma, nodular melanoma, acrallentiginous melanoma, and lentigo maligna (melanoma). Squamous cellscarcinomas include, but are not limited to head and neck squamous cellcarcinoma, and squamous cell cancers of the lung.

Compositions comprising the endoplasmin antibodies can also be used toprevent metastasis or decrease the number of micrometastases, such asmicrometastases to regional lymph nodes. Immunoconjugates comprising theendoplasmin antibodies also can be used to treat a patient diagnosedwith cancer. The human monoclonal antibodies can also be used todiagnose cancer in a subject, including the detection of a metastasis.For example, the human monoclonal antibodies can be contacted with asample from the patient, such as a serum sample, to detect elevatedlevels of endoplasmin. The antibodies and compositions provided hereincan also be used to detect cancer in a subject or to confirm thediagnosis of cancer in a patient.

Disclosed herein are fully human monoclonal antibodies that specificallybind human endoplasmin and functional fragments thereof (antigen-bindingfragments) that specifically bind endoplasmin. A major limitation in theclinical use of mouse monoclonal antibodies is the development of ahuman anti-murine antibody (HAMA) response in the patients receiving thetreatments. The HAMA response can involve allergic reactions and anincreased rate of clearance of the administered antibody from the serum.Various types of modified monoclonal antibodies have been developed tominimize the HAMA response while trying to maintain the antigen bindingaffinity of the parent monoclonal antibody. One type of modifiedmonoclonal antibody is a human-mouse chimera in which a murineantigen-binding variable region is coupled to a human constant domain(Morrison and Schlom, Important Advances in Oncology, Rosenberg, S. A.(Ed.), 1989). A second type of modified monoclonal antibody is thecomplementarity determining region (CDR)-grafted, or humanized,monoclonal antibody (Winter and Harris, Immunol. Today 14:243-246,1993). However, the antibodies disclosed herein are fully human; boththe framework region and the CDRs are derived from human sequences.Thus, a HAMA is not induced when these antibodies are administered to ahuman subject.

In some embodiments, the human monoclonal antibody or antigen-bindingfragment thereof comprises at least a portion of the variable chain ofthe heavy chain amino acid sequence set forth as SEQ ID NO: 1 andspecifically binds endoplasmin. For example, the human monoclonalantibody can include the SDRs (specificity determining residues, theCDRs, or the variable region. In the amino acid sequence shown below,the constant region is in bold, and the CDRs are underlined:

  (SEQ ID NO: 1)

VTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMetHEALHNHYT QKSLSLSPGK

In some embodiments, the human monoclonal antibody, or antigen-bindingfragment thereof, comprises at least a portion of the heavy chain aminoacid sequence set forth as SEQ ID NO: 1 and specifically bindsendoplasmin. In some examples, at least one of the CDRs of the lightchain of the antibody comprises one or more of the amino acid sequencesset forth as amino acids 26-33 of SEQ ID NO: 1 (CDR1), amino acids 51-58of SEQ ID NO: 1 (CDR2), and amino acids 97-103 of SEQ ID NO: 1 (CDR3).In additional examples, the heavy chain of the antibody comprises theamino acid sequence set forth as amino acids 26-33 of SEQ ID NO: 1(CDR1), amino acids 51-58 of SEQ ID NO: 1 (CDR2), and amino acids 97-103of SEQ ID NO: 1 (CDR3). In some examples, the variable region of theheavy chain of the antibody can include, or consist of, amino acids1-113 of SEQ ID NO: 1. The heavy chain of the antibody can include, orconsist of, SEQ ID NO: 1.

In some embodiments, the human monoclonal antibody, or antigen-bindingfragment thereof, comprises at least a portion of the variable region ofthe light chain amino acid sequence set forth as SEQ ID NO: 2 andspecifically binds endoplasmin. In the amino acid sequence shown below,the constant region is in bold, and the CDRs are underlined:

(SEQ ID NO: 2)

FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGECIn some examples, at least one of the CDRs of the light chain of theantibody comprises one or more of the amino acid sequences set forth asamino acids 27-32 of SEQ ID NO: 2 (CDR1), amino acids 50-52 of SEQ IDNO: 2 (CDR2), and amino acids 89-97 of SEQ ID NO: 2 (CDR3). Inadditional examples, the light chain of the antibody comprises aminoacids amino acids 27-32 of SEQ ID NO: 2 (CDR1), amino acids 50-52 of SEQID NO: 2 (CDR2), and amino acids 89-97 of SEQ ID NO: 2 (CDR3). Thevariable region of the light chain of the antibody can include, orconsist of, amino acids 1-107 of SEQ ID NO: 2. The light chain of theantibody can include, or consist of, SEQ ID NO: 2.

In some embodiments, the human monoclonal antibody is labeled. In someexamples, the label is a fluorescence, enzymatic, or radioactive label.

The monoclonal antibody can be of any isotype. The monoclonal antibodycan be, for example, an IgA, IgM or an IgG antibody, such as IgG₁ or anIgG₂. The class of an antibody that specifically binds endoplasmin canbe switched with another. In one aspect, a nucleic acid moleculeencoding V_(L) or V_(H) is isolated using methods well-known in the art,such that it does not include any nucleic acid sequences encoding theconstant region of the light or heavy chain, respectively. The nucleicacid molecule encoding V_(L) or V_(H) is then operatively linked to anucleic acid sequence encoding a C_(L) or C_(H) from a different classof immunoglobulin molecule. This can be achieved using a vector ornucleic acid molecule that comprises a C_(L) or C_(H) chain, as known inthe art. For example, an antibody that specifically binds endoplasminthat was originally IgM may be class switched to an IgG. Class switchingcan be used to convert one IgG subclass to another, such as from IgG1 toIgG₂.

Fully human monoclonal antibodies include human framework regions. Thehuman framework regions can include the framework regions disclosed inone or both of SEQ ID NO: 1 or SEQ ID NO: 2 (these sequences include CDRsequences as well as framework sequences). However, the frameworkregions can be from another source. Additional examples of frameworksequences that can be used include the amino acid framework sequences ofthe heavy and light chains disclosed in PCT Publication No. WO2006/074071 (see, for example, SEQ ID NOs: 1-16), which is hereinincorporated by reference.

Antibody fragments are encompassed by the present disclosure, such asFab, F(ab′)₂, scFv and Fv which include a heavy chain and light chainvariable region and bind the epitopic determinant on endoplasmin. Theseantibody fragments retain the ability to specifically bind with theantigen, namely human endoplasmin, and thus are antigen-bindingfragments. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988).

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of asingle chain antibody (scFV₂), are also contemplated.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced that specifically bind human endoplasmin. Suchconservative variants employed in antibody fragments, such as dsFvfragments or in scFv fragments, will retain critical amino acid residuesnecessary for correct folding and stabilizing between the V_(H) and theV_(L) regions, and will retain the charge characteristics of theresidues in order to preserve the low pI and low toxicity of themolecules. Amino acid substitutions (such as at most one, at most two,at most three, at most four, or at most five amino acid substitutions)can be made in the V_(H) and the V_(L) regions to increase yield.Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

IV. Use in Therapeutic and Diagnostic Moieties

The human monoclonal antibodies, or functional fragments thereof thatspecifically bind human endoplasmin can be used in therapeutic anddiagnostic methods, such as for the treatment and detection of melanoma,breast cancer, head and neck squamous cell carcinoma, renal cancer, lungcancer, glioma, bladder cancer, ovarian cancer or pancreatic cancer. Fortherapeutic use, the methods can include administering to subject atherapeutically effective amount of an antibody that specifically bindsendoplasmin, or an antigen-binding fragment thereof, such as for thetreatment of melanoma, breast cancer, head and neck squamous cellcarcinoma, renal cancer, lung cancer, glioma, bladder cancer, ovariancancer or pancreatic cancer, such as pancreatic adenocarcinoma.

In several embodiments, the human monoclonal antibodies orantigen-binding fragments thereof described herein can be conjugated toa therapeutic agent. Immunoconjugates include, but are not limited to,molecules in which there is a covalent linkage of a therapeutic agent toan antibody. A therapeutic agent is an agent with a particularbiological activity directed against a particular target molecule or acell bearing a target molecule. One of skill in the art will appreciatethat therapeutic agents can include various drugs such as vinblastine,daunomycin and the like, cytotoxins such as native or modifiedPseudomonas exotoxin or Diphtheria toxin, encapsulating agents (such asliposomes) which themselves contain pharmacological compositions,radioactive agents such as ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S and other labels,target moieties and ligands.

Toxins can be employed with the endoplasmin-specific human monoclonalantibodies, and antigen-binding fragments thereof, that are describedherein, to produce immunotoxins. Exemplary toxins include ricin, abrin,diphtheria toxin and subunits thereof, as well as botulinum toxins Athrough F. These toxins are readily available from commercial sources(for example, Sigma Chemical Company, St. Louis, Mo.). Contemplatedtoxins also include variants of the toxins described herein (see, forexample, see, U.S. Pat. Nos. 5,079,163 and 4,689,401). In oneembodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Pat. No.5,602,095). As used herein “Pseudomonas exotoxin” refers to afull-length native (naturally occurring) PE or a PE that has beenmodified. Such modifications can include, but are not limited to,elimination of domain Ia, various amino acid deletions in domains Ib, IIand III, single amino acid substitutions and the addition of one or moresequences at the carboxyl terminus (for example, see Siegall et al., J.Biol. Chem. 264:14256-14261, 1989). In one embodiment, the cytotoxicfragment of PE retains at least 50%, at least 75%, at least 90%, or atleast 95% of the cytotoxicity of native PE. In some examples, thecytotoxic fragment is more toxic than native PE.

Native Pseudomonas exotoxin A (PE) is an extremely active monomericprotein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa,which inhibits protein synthesis in eukaryotic cells. The method of PEaction is inactivation of the ADP-ribosylation of elongation factor 2(EF-2). The exotoxin contains three structural domains that act inconcert to cause cytotoxicity. Domain 1a mediates cell binding. DomainII is responsible for translocation into the cytosol and domain IIImediates ADP ribosylation of elongation factor 2. The function of domainIb is unknown. PE employed with the monoclonal antibodies describedherein can include the native sequence, cytotoxic fragments of thenative sequence, and conservatively modified variants of native PE andits cytotoxic fragments. Cytotoxic fragments of PE include those whichare cytotoxic with or without subsequent proteolytic or other processingin the target cell. Cytotoxic fragments of PE include PE40, PE38, andPE35. For additional description of PE and variants thereof, see forexample, U.S. Pat. Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039;5,821,238; and 5,854,044; PCT Publication No. WO 99/51643; Pai et al.,Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol.Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta1333:C1-C6, 1997, each of which is herein incorporated by reference.

The antibodies and antigen-binding fragments thereof described hereincan also be used to target any number of different diagnostic ortherapeutic compounds to cells expressing endoplasmin on their surface.Thus, an antibody of the present disclosure can be attached directly orvia a linker to a drug that is to be delivered directly to cellsexpressing cell-surface endoplasmin. Therapeutic agents include suchcompounds as nucleic acids, proteins, peptides, amino acids orderivatives, glycoproteins, radioisotopes, lipids, carbohydrates, orrecombinant viruses. Nucleic acid therapeutic and diagnostic moietiesinclude antisense nucleic acids, derivatized oligonucleotides forcovalent cross-linking with single or duplex DNA, and triplex formingoligonucleotides.

Alternatively, the molecule linked to an anti-endoplasmin antibody canbe an encapsulation system, such as a liposome or micelle that containsa therapeutic composition such as a drug, a nucleic acid (for example,an antisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art (see, for example, U.S. Pat. No. 4,957,735;Connor et al., Pharm. Ther. 28:341-365, 1985).

The antibodies disclosed herein can be conjugated to an additionaltherapeutic agent and/or can be used in conjunction with an additionagent, using sequential or simultaneous administration. The choice of aparticular therapeutic agent depends on the particular target moleculeor cell, and the desired biological effect. Thus, for example, thetherapeutic agent can be a cytotoxin that is used to bring about thedeath of a particular target cell. Conversely, where it is desired toinvoke a non-lethal biological response, the therapeutic agent can beconjugated to a non-lethal pharmacological agent or a liposomecontaining a non-lethal pharmacological agent.

The therapeutic agent can also be a cytokine or a chemokine. A“cytokine” is class of proteins or peptides released by one cellpopulation which act on another cell as intercellular mediators.Cytokines can act as an immune-modulating agent. Examples of cytokinesinclude lymphokines, monokines, growth factors and traditionalpolypeptide hormones. Thus, embodiments utilize an interferon (e.g.,IFN-α, IFN-β, and IFN-γ); tumor necrosis factor super family (TNFSF)member; human growth hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; follicle stimulating hormone (FSH); thyroid stimulatinghormone (TSH); luteinizing hormone (LH); hepatic growth factor;prostaglandin, fibroblast growth factor; prolactin; placental lactogen,OB protein; TNF-α; TNF-β; integrin; thrombopoietin (TPO); a nerve growthfactor such as NGF-β; platelet-growth factor; TGF-α; TGF-β; insulin-likegrowth factor-I and -II; erythropoietin (EPO); colony stimulatingfactors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); aninterleukin (IL-1 to IL-21), kit-ligand or FLT-3, angiostatin,thrombospondin, or endostatin. These cytokine include proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

Chemokines can also be conjugated to the antibodies disclosed herein.Chemokines are a superfamily of small (approximately about 4 to about 14kDa), inducible and secreted pro-inflammatory cytokines that actprimarily as chemoattractants and activators of specific leukocyte cellsubtypes. Chemokine production is induced by inflammatory cytokines,growth factors and pathogenic stimuli. The chemokine proteins aredivided into subfamilies (alpha, beta, and delta) based on conservedamino acid sequence motifs and are classified into four highly conservedgroups—CXC, CC, C and CX3C, based on the position of the first twocysteines that are adjacent to the amino terminus. To date, more than 50chemokines have been discovered and there are at least 18 humanseven-transmembrane-domain (7TM) chemokine receptors. Chemokines of useinclude, but are not limited to, RANTES, MCAF, MCP-1, and fractalkine.

The therapeutic agent can be a chemotherapeutic agent. In oneembodiment, the chemotherapeutic agent is radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer L., Berkery R. (eds): OncologyPocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995;Fischer D S, Knobf M F, Durivage H J (eds): The Cancer ChemotherapyHandbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Chemotherapeuticagents include those known by those skilled in the art, including butnot limited to: 5-fluorouracil (5-FU), azathioprine, cyclopamine,cyclophosphamide, antimetabolites (such as Fludarabine), antineoplastics(such as etoposide, doxorubicin, methotrexate, and vincristine),carboplatin, cis-platinum, dacarbazine, temozolomide, PARP inhibitorsand the taxanes, such as taxol. Rapamycin has also been used as achemotherapeutic agent.

Effector molecules, such as, but not limited to, radionucleotide,cytokines, chemokines and chemotherapeutic agents, can be linked to anantibody of interest using any number of means known to those of skillin the art. Both covalent and noncovalent attachment means may be used.The procedure for attaching an effector molecule to an antibody variesaccording to the chemical structure of the effector. Polypeptidestypically contain a variety of functional groups; such as carboxylicacid (COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on an antibodyto result in the binding of the effector molecule. Alternatively, theantibody is derivatized to expose or attach additional reactivefunctional groups. The derivatization may involve attachment of any of anumber of linker molecules such as those available from Pierce ChemicalCompany, Rockford, Ill. The linker can be any molecule used to join theantibody to the effector molecule. The linker is capable of formingcovalent bonds to both the antibody and to the effector molecule.Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. Where theantibody and the effector molecule are polypeptides, the linkers may bejoined to the constituent amino acids through their side groups (such asthrough a disulfide linkage to cysteine) or to the alpha carbon aminoand carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (such as enzymes or fluorescent molecules) drugs,toxins, polypeptides and other agents to antibodies one skilled in theart will be able to determine a suitable method for attaching a givenagent to an antibody or other polypeptide.

The antibodies or antibody fragments that specifically bind endoplasmindisclosed herein can be derivatized or linked to another molecule (suchas another peptide or protein). In general, the antibodies or portionthereof is derivatized such that the binding to endoplasmin is notaffected adversely by the derivatization or labeling. For example, theantibody can be functionally linked (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (for example, a bispecificantibody or a diabody), a detection agent, a pharmaceutical agent,and/or a protein or peptide that can mediate associate of the antibodyor antibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

Methods for the detection of endoplasmin are provided herein, includingmethods for detecting cells expressing endoplasmin, such as melanoma,breast cancer, head and neck squamous cell carcinoma, renal cancer, lungcancer, glioma, bladder cancer, ovarian cancer or pancreatic cancercells. These methods can include contacting a sample from a subject withan antibody that specifically binds endoplasmin, or antigen-bindingfragment thereof, as disclosed herein. The methods can be used to detecta primary tumor, or can be used to detect metastases.

In some embodiments, methods are provided for detecting cancer orconfirming the diagnosis of cancer in a subject. The method includescontacting a biological sample from the subject with an isolatedantibody that specifically binds endoplasmin or antigen-biding fragmentthereof and detecting binding of the isolated human monoclonal antibody,or antigen-binding fragment thereof to the sample. An increase inbinding of the isolated human monoclonal antibody, or antigen-bindingfragment thereof to the sample as compared to binding of the isolatedhuman monoclonal antibody, or antigen-binding fragment thereof, to acontrol sample detects cancer in the subject or confirms the diagnosisof cancer in the subject. The control can be a sample from a subjectknown not to have cancer, or a standard value.

The sample can be any sample, including, but not limited to, tissue frombiopsies, autopsies and pathology specimens. Biological samples alsoinclude sections of tissues, for example, frozen sections taken forhistological purposes. Biological samples further include body fluids,such as blood, serum, plasma, sputum, and spinal fluid.

In some embodiments, the human antibody that specifically bindsendoplasmin (the first antibody) is unlabeled and a second antibody orother molecule that can bind the antibody that specifically bindsendoplasmin is labeled. As is well known to one of skill in the art, asecond antibody is chosen that is able to specifically bind the specificspecies and class of the first antibody. For example, if the firstantibody is a human IgG, then the secondary antibody may be ananti-human-IgG. Other molecules that can bind to antibodies include,without limitation, Protein A and Protein G, both of which are availablecommercially.

A human antibody that specifically binds endoplasmin or antigen bindingfragment thereof can be labeled with a detectable moiety. Usefuldetection agents include fluorescent compounds, including fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein (GFP), Yellow fluorescent protein(YFP). An antibody can also be labeled with enzymes that are useful fordetection, such as horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase, glucose oxidase and the like. When an antibody islabeled with a detectable enzyme, it can be detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody may also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Itshould be noted that the avidin itself can be labeled with an enzyme ora fluorescent label.

An antibody may be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes. Forinstance, the radiolabel may be used to detect endoplasmin by x-ray,emission spectra, magnetic resonance imaging (MRI), commuted tomography(CT) scan, positron emission tomography (PET), or other diagnostictechniques. Examples of labels for polypeptides include, but are notlimited to, the following radioisotopes or radionucleotides: ³⁵S, ¹¹C,¹³N, ¹⁵O, ¹⁸F, ^(99m)Tc, ¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

Antibodies described herein can also be covalently or non-covalentlylinked to a detectable label. Detectable labels suitable for such useinclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels include magnetic beads, fluorescent dyes (for example,fluorescein isothiocyanate, Texas red, rhodamine, green fluorescentprotein, and the like), radiolabels (for example, ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (such as horseradish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (such as polystyrene,polypropylene, latex, and the like) beads. These antibodies can be usedin a variety of immunoassays, including Fluorescence activated cellssorting (FACS), immunohistochemistry, radioimmune assays (RIAs), andenzyme-linked immunosorbant assays (ELISA).

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

In one embodiment, a kit is provided for detecting endoplasmin in abiological sample, such as a blood sample. Kits for detecting apolypeptide will typically comprise a human antibody that specificallybinds endoplasmin, such as any of the antibodies disclosed herein. Insome embodiments, an antibody fragment, such as an Fv fragment isincluded in the kit. For in vivo uses, the antibody can be a scFvfragment. In a further embodiment, the antibody is labeled (for example,with a fluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds endoplasmin. Theinstructional materials may be written, in an electronic form (such as acomputer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting endoplasmin in a biological samplegenerally includes the steps of contacting the biological sample with anantibody which specifically reacts, under immunologically reactiveconditions, to endoplasmin. The antibody is allowed to specifically bindunder immunologically reactive conditions to form an immune complex, andthe presence of the immune complex (bound antibody) is detected directlyor indirectly.

V. Endoplasmin Antibody Polynucleotides and Polypeptides

Nucleic acid molecules (also referred to as polynucleotides) encodingthe polypeptides provided herein (including, but not limited toantibodies, antigen-binding fragments thereof, immunoconjugates andfusion proteins) can readily be produced by one of skill in the art,using the amino acid sequences provided herein, sequences available inthe art, and the genetic code. In addition, one of skill can readilyconstruct a variety of clones containing functionally equivalent nucleicacids, such as nucleic acids which differ in sequence but which encodethe same effector molecule or antibody sequence. Thus, nucleic acidsencoding antibodies, conjugates and fusion proteins are provided herein.

In some embodiments, the endoplasmin human monoclonal antibodies have aV_(H) domain encoded by a nucleotide sequence comprising SEQ ID NO: 3.In some embodiments, the endoplasmin human monoclonal antibodies have aV_(L) domain encoded by the nucleotide sequence comprising SEQ ID NO: 4.Exemplary nucleic acid sequences are provided below:

W9 VH sequence (hIgG1 Constant Heavy region in bold):

(SEQ ID NO: 3) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACTAGCTATGCTATGCATTGGGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCTGGCAATGGTAACACAAAATATTCACAGAAGTTCCAGGGCAGAGTCACCATTACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCCGTGTATTACTGTGCAAGGGCCCATTTTGACTACTGGGGCCAAGGTACCCTGGTCACCGTCTCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC CCTGCCCCCATCCCGGGA G GAGA TGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAATGAW9 VL sequence (Human Kappa Constant Light region in bold):

(SEQ ID NO: 4) GAAATTGAGCTCACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTACTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCAACGTTCGGCCAAGGGACCAAGGTGGAGATCAAAACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTTAG

Nucleic acid sequences encoding the human antibodies that specificallybind endoplasmin, or antigen-binding fragments thereof that specificallybind endoplasmin, can be prepared by any suitable method including, forexample, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids encoding human antibodies that specifically bindendoplasmin, or functional fragments thereof that specifically bindendoplasmin, can be prepared by cloning techniques. Examples ofappropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Sambrook et al., supra, Berger and Kimmel (eds.), supra, andAusubel, supra. Product information from manufacturers of biologicalreagents and experimental equipment also provide useful information.Such manufacturers include the SIGMA Chemical Company (Saint Louis,Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway,N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem GenesCorp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill

Nucleic acids encoding native effector molecule (EM) or anti-endoplasminantibodies can be modified to form the EM, antibodies, orimmunoconjugates of the present disclosure. Modification bysite-directed mutagenesis is well known in the art. Nucleic acids canalso be prepared by amplification methods. Amplification methods includepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (3SR). A wide variety of cloning methods,host cells, and in vitro amplification methodologies are well known topersons of skill

In one embodiment, immunoconjugates are prepared by inserting the cDNAwhich encodes a human endoplasmin-specific monoclonal antibody orantigen-binding fragment thereof into a vector which comprises the cDNAencoding the EM. The insertion is made so that the antibody and the EMare read in frame, that is in one continuous polypeptide which containsa functional antibody region and a functional EM region. In oneembodiment, cDNA encoding an EM, label or enzyme is ligated to anantibody so that the EM, label or enzyme is located at the carboxylterminus of the antibody. In another embodiment, the EM, label or enzymeis located at the amino terminus of the antibody. In a another example,cDNA encoding the EM, label or enzyme is ligated to a heavy chainvariable region of an antibody, so that the EM, label or enzyme islocated at the carboxyl terminus of the heavy chain variable region. Theheavy chain-variable region can subsequently be ligated to a light chainvariable region of the antibody using disulfide bonds. In a yet anotherexample, cDNA encoding an EM, label or enzyme is ligated to a lightchain variable region of an antibody, so that the EM, label or enzyme islocated at the carboxyl terminus of the light chain variable region. Thelight chain-variable region can subsequently be ligated to a heavy chainvariable region of the antibody using disulfide bonds.

Once the nucleic acids encoding an EM, anti-endoplasmin antibody,antigen-binding fragment thereof, or an immunoconjugate, are isolatedand cloned, the desired protein can be expressed in a recombinantlyengineered cell such as bacteria, plant, yeast, insect and mammaliancells. It is expected that those of skill in the art are knowledgeablein the numerous expression systems available for expression of proteinsincluding E. coli, other bacterial hosts, yeast, and various highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart. Hybridomas expressing the antibodies of interest are alsoencompassed by this disclosure.

The expression of nucleic acids encoding the isolated antibodies andantibody fragments described herein can be achieved by operably linkingthe DNA or cDNA to a heterologous promoter (which is either constitutiveor inducible), followed by incorporation into an expression cassette.The cassettes can be suitable for replication and integration in eitherprokaryotes or eukaryotes. Typical expression cassettes contain specificsequences useful for regulation of the expression of the DNA encodingthe protein. For example, the expression cassettes can includeappropriate promoters, enhancers, transcription and translationterminators, initiation sequences, a start codon (i.e., ATG) in front ofa protein-encoding gene, splicing signal for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons.

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.For E. coli, this includes a promoter such as the T7, trp, lac, orlambda promoters, a ribosome binding site, and preferably atranscription termination signal. For eukaryotic cells, the controlsequences can include a promoter and/or an enhancer derived from, forexample, an immunoglobulin gene, SV40 or cytomegalovirus, and apolyadenylation sequence, and can further include splice donor andacceptor sequences. The cassettes can be transferred into the chosenhost cell by well-known methods such as transformation orelectroporation for E. coli and calcium phosphate treatment,electroporation or lipofection for mammalian cells. Cells transformed bythe cassettes can be selected by resistance to antibiotics conferred bygenes contained in the cassettes, such as the amp, gpt, neo and hyggenes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or antigen-binding fragment thereof, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein (see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein (i.e., a human endoplasmin-specific monoclonal antibodyor an immunoconjugate comprising the antibody) without diminishing itsbiological activity. Some modifications can be made to facilitate thecloning, expression, or incorporation of the targeting molecule into afusion protein. Such modifications are well known to those of skill inthe art and include, for example, termination codons, a methionine addedat the amino terminus to provide an initiation, site, additional aminoacids placed on either terminus to create conveniently locatedrestriction sites, or additional amino acids (such as poly His) to aidin purification steps. In addition to recombinant methods, theimmunoconjugates, effector moieties, and antibodies of the presentdisclosure can also be constructed in whole or in part using standardpeptide synthesis well known in the art.

Once expressed, the recombinant immunoconjugates, antibodies, and/oreffector molecules can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, and the like (see, generally, R. Scopes, PROTEINPURIFICATION, Springer-Verlag, N.Y., 1982). The antibodies,immunoconjugates and effector molecules need not be 100% pure. Oncepurified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand antigen-binding fragments thereof that are disclosed herein can alsobe constructed in whole or in part using standard peptide synthesis.Solid phase synthesis of the polypeptides of less than about 50 aminoacids in length can be accomplished by attaching the C-terminal aminoacid of the sequence to an insoluble support followed by sequentialaddition of the remaining amino acids in the sequence. Techniques forsolid phase synthesis are described by Barany & Merrifield, ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem.Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase PeptideSynthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Proteins ofgreater length may be synthesized by condensation of the amino andcarboxyl termini of shorter fragments. Methods of forming peptide bondsby activation of a carboxyl terminal end (such as by the use of thecoupling reagent N,N′-dicylohexylcarbodimide) are well known in the art.

VI. Compositions and Therapeutic Methods

Compositions are provided herein that include a carrier and one or moreof the antibodies that specifically bind endoplasmin, or antigen-bindingfragment thereof that specifically binds endoplasmin. Compositionscomprising immunoconjugates or immunotoxins are also provided. Thecompositions can be prepared in unit dosage forms for administration toa subject. The amount and timing of administration are at the discretionof the treating physician to achieve the desired purposes. The antibodycan be formulated for systemic or local (such as intra-tumor)administration. In one example, the antibody that specifically bindsendoplasmin is formulated for parenteral administration, such asintravenous administration.

The compositions for administration can include a solution of theantibody that specifically binds endoplasmin (or a functional fragmentthereof that specifically binds endoplasmin) dissolved in apharmaceutically acceptable carrier, such as an aqueous carrier. Avariety of aqueous carriers can be used, for example, buffered salineand the like. These solutions are sterile and generally free ofundesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of antibody in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the subject's needs.

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg of antibody per subject per day. Dosagesfrom 0.1 up to about 100 mg per subject per day may be used,particularly if the agent is administered to a secluded site and notinto the circulatory or lymph system, such as into a body cavity or intoa lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa.(1995).

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.5 to 15 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of RITUXAN® in 1997. Antibodies can be administeredby slow infusion, rather than in an intravenous push or bolus. In oneexample, a higher loading dose is administered, with subsequent,maintenance doses being administered at a lower level. For example, aninitial loading dose of 4 mg/kg may be infused over a period of some 90minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kginfused over a 30 minute period if the previous dose was well tolerated.

The antibody that specifically binds endoplasmin (or antigen-bindingfragment thereof or immunoconjugate thereof) can be administered to slowor inhibit the growth of cells, such as cancer cells. In theseapplications, a therapeutically effective amount of an antibody isadministered to a subject in an amount sufficient to inhibit growth,replication or metastasis of cancer cells, or to inhibit a sign or asymptom of the cancer. In some embodiments, the antibodies areadministered to a subject to inhibit or prevent the development ofmetastasis, or to decrease the size or number of metasases, such asmicrometastases, for example micrometastases to the regional lymph nodes(Goto et al., Clin. Cancer Res. 14(11):3401-3407, 2008).

Suitable subjects may include those diagnosed with a cancer thatexpresses endoplasmin, such as, but not limited to, melanoma, breastcancer, head and neck squamous cell carcinoma, renal cancer, lungcancer, glioma, bladder cancer, ovarian cancer or pancreatic cancer. Atherapeutically effective amount of a human endoplasmin-specificantibody will depend upon the severity of the disease and the generalstate of the patient's health. A therapeutically effective amount of theantibody is that which provides either subjective relief of a symptom(s)or an objectively identifiable improvement as noted by the clinician orother qualified observer. These compositions can be administered inconjunction with another chemotherapeutic agent, either simultaneouslyor sequentially.

Many chemotherapeutic agents are presently known in the art. In oneembodiment, the chemotherapeutic agents is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, anti-survival agents,biological response modifiers, anti-hormones, e.g. anti-androgens, andanti-angiogenesis agents.

Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2)inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II(cyclooxygenase II) inhibitors, can be used in conjunction with acompound of the invention. Examples of useful COX-II inhibitors includeCELEBREX™ (alecoxib), valdecoxib, and rofecoxib. Examples of usefulmatrix metalloproteinase inhibitors are described in PCT Publication No.WO 96/33172 (published Oct. 24, 1996), PCT Publication No. WO 96/27583(published Mar. 7, 1996), European Patent Application No. 97304971.1(filed Jul. 8, 1997), European Patent Application No. 99308617.2 (filedOct. 29, 1999), PCT Publication No. WO 98/07697 (published Feb. 26,1998), PCT Publication No WO 98/03516 (published Jan. 29, 1998), PCTPublication No WO 98/34918 (published Aug. 13, 1998), PCT Publication NoWO 98/34915 (published Aug. 13, 1998), PCT Publication No WO 98/33768(published Aug. 6, 1998), PCT Publication No WO 98/30566 (published Jul.16, 1998), European Patent Publication 606,046 (published Jul. 13,1994), European Patent Publication 931,788 (published Jul. 28, 1999),PCT Publication No WO 90/05719 (published May 31, 1990), PCT PublicationNo WO 99/52910 (published Oct. 21, 1999), PCT Publication No WO 99/52889(published Oct. 21, 1999), PCT Publication No WO 99/29667 (publishedJun. 17, 1999), PCT International Application No. PCT/IB98/01113 (filedJul. 21, 1998), European Patent Application No. 99302232.1 (filed Mar.25, 1999), U.S. Pat. No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No.5,861,510 (issued Jan. 19, 1999), and European Patent Publication780,386 (published Jun. 25, 1997). In one example, the MMP inhibitors donot induce arthralgia upon administration. In another example, the MMPinhibitor selectively inhibits MMP-2 and/or MMP-9 relative to the othermatrix-metalloproteinases (such as MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specificexamples of MMP inhibitors of use are AG-3340, RO 32-3555, RS 13-0830,34[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino-1-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxaicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofsaid compounds.

The antibodies that specifically bind endoplasmin can also be used withsignal transduction inhibitors, such as agents that can inhibit EGF-R(epidermal growth factor receptor) responses, such as EGF-R antibodies,EGF antibodies, and molecules that are EGF-R inhibitors; VEGF (vascularendothelial growth factor) inhibitors, such as VEGF receptors andmolecules that can inhibit VEGF; and erbB2 receptor inhibitors, such asorganic molecules or antibodies that bind to the erbB2 receptor, forexample, HERCEPTIN™ (Genentech, Inc.). EGF-R inhibitors are describedin, for example in PCT Publication Nos. WO 95/19970 (published Jul. 27,1995), WO 98/14451 (published Apr. 9, 1998), WO 98/02434 (published Jan.22, 1998), and U.S. Pat. No. 5,747,498 (issued May 5, 1998).EGFR-inhibiting agents also include, but are not limited to, themonoclonal antibodies C225 and anti-EGFR 22Mab (ImClone SystemsIncorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA),EMD-5590 (Merck KgaA), MDX-447/H-477 (Medarex Inc. and Merck KgaA), andthe compounds ZD-1834, ZD-1838 and ZD-1839 (AstraZeneca), PKI-166(Novartis), PKI-166/CGP-75166 (Novartis), PTK 787 (Novartis), CP 701(Cephalon), leflunomide (Pharmacia/Sugen), C1-1033 (Warner Lambert ParkeDavis), C1-1033/PD 183,805 (Warner Lambert Parke Davis), CL-387,785(Wyeth-Ayerst), BBR-1611 (Boehringer Mannheim GmbH/Roche), Naamidine A(Bristol Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (BoehringerIngelheim), OLX-103 (Merck & Co.), VRCTC-310 (Ventech Research), EGFfusion toxin (Seragen Inc.), DAB-389 (Seragen/Lilgand), ZM-252808(Imperial Cancer Research Fund), RG-50864 (INSERM), LFM-A12 (ParkerHughes Cancer Center), WHI-P97 (Parker Hughes Cancer Center), GW-282974(Glaxo), KT-8391 (Kyowa Hakko) and EGF-R Vaccine (York Medical/Centro deImmunologia Molecular (CIM)).

VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.), SH-268(Schering), and NX-1838 (NeXstar) can also be used in conjunction withan antibody that specifically binds endoplasmin. VEGF inhibitors aredescribed in, for example in PCT Publication No. WO 99/24440 (publishedMay 20, 1999), PCT International Application PCT/IB99/00797 (filed May3, 1999), PCT Publication No. WO 95/21613 (published Aug. 17, 1995), PCTPublication No. WO 99/61422 (published Dec. 2, 1999), U.S. Pat. No.5,834,504 (issued Nov. 10, 1998), PCT Publication No. WO 98/50356(published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar. 16,1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No.5,792,783 (issued Aug. 11, 1998), PCT Publication No. WO 99/10349(published Mar. 4, 1999), PCT Publication No. WO 97/32856 (publishedSep. 12, 1997), PCT Publication No. WO 97/22596 (published Jun. 26,1997), PCT Publication No. WO 98/54093 (published Dec. 3, 1998), PCTPublication No. WO 98/02438 (published Jan. 22, 1998), WO 99/16755(published Apr. 8, 1999), and PCT Publication No. WO 98/02437 (publishedJan. 22, 1998). Other examples of some specific VEGF inhibitors areIM862 (Cytran Inc.); anti-VEGF monoclonal antibody of Genentech, Inc.;and angiozyme, a synthetic ribozyme from Ribozyme and Chiron. These andother VEGF inhibitors can be used in conjunction with an antibody thatspecifically binds endoplasmin.

ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome pic), andthe monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc.) and 2B-1(Chiron), can furthermore be combined with the compound of theinvention, for example those indicated in PCT Publication No. WO98/02434 (published Jan. 22, 1998), PCT Publication No. WO 99/35146(published Jul. 15, 1999), PCT Publication No. WO 99/35132 (publishedJul. 15, 1999), PCT Publication No. WO 98/02437 (published Jan. 22,1998), PCT Publication No. WO 97/13760 (published Apr. 17, 1997), PCTPublication No. WO 95/19970 (published Jul. 27, 1995), U.S. Pat. No.5,587,458 (issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305 (issuedMar. 2, 1999). ErbB2 receptor inhibitors of use are also described inU.S. Provisional Application No. 60/117,341, filed Jan. 27, 1999, and inU.S. Provisional Application No. 60/117,346, filed Jan. 27, 1999.

The antibodies that specifically bind endoplasmin (or an antigen-bindingfragment thereof) can be used with, and/or conjugated to, a cytokine ora chemokine, or can be conjugated to a cytokine or a chemokine.Exemplary cytokines include, but are not limited to, interferons (IFNs),such as IFN-α, IFN-β, and IFN-γ); tumor necrosis factor super family(TNFSF) members; human growth hormone; thyroxine; insulin; proinsulin;relaxin; prorelaxin; follicle stimulating hormone (FSH); thyroidstimulating hormone (TSH); luteinizing hormone (LH); hepatic growthfactor; prostaglandin, fibroblast growth factor; prolactin; placentallactogen, OB protein; tumor necrosis faction (TNF)-α; TNF-β; integrin;thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-13;platelet-growth factor; transforming growth factor (TGF)-α; TGF-β;insulin-like growth factor-I and -II; erythropoietin (EPO); colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (IL-1 to IL-21), kit-ligand or FLT-3, angiostatin,thrombospondin, and endostatin. Suitable chemokines include, but are notlimited to, RANTES, MCAF, MCP-1, and fractalkine.

For the treatment of cancer, such as melanoma, the antibodies disclosedherein can be used with surgical treatment, or with another therapeuticincluding dacarbazine (also termed DTIC), temozolomide, PARP inhibitorsor interleukin-2 (IL-2) or interferon, such as interferon (IFN), orcombinations of these agents. For the treatment of a superficialmelanoma, the antibodies can be used in conjunction with Imiquimod. Forthe treatment of head and neck squamous cell carcinoma, the antibodiesprovided herein can be used in conjunction with surgery, radiationtherapy, chemotherapy, other antibodies (such as cetuximab andbevacizumab) or small-molecule therapeutics (such as erlotinib).

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of at least one of the antibodies (or antigen-binding fragmentsthereof) disclosed herein to effectively treat the patient. The dosagecan be administered once but may be applied periodically until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. In one example, a dose of the antibody isinfused for thirty minutes every other day. In this example, about oneto about ten doses can be administered, such as three or six doses canbe administered every other day. In a further example, a continuousinfusion is administered for about five to about ten days. The subjectcan be treated at regular intervals, such as monthly, until a desiredtherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the patient.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein, such as a cytotoxin or a drug, as a central core.In microspheres the therapeutic is dispersed throughout the particle.Particles, microspheres, and microcapsules smaller than about 1 μm aregenerally referred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992) both of which are incorporated herein by reference.

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496).

Fully human monoclonal antibodies that specifically bind endoplasmin, ora antigen-binding fragment thereof, covalently linked to an effectormolecule can be used for a variety of purposes, including forradioimmunotherapy or radioimmunoguided surgery. For example, anendoplasmin antibody can be linked to a radioactive isotope and used inimmunotherapy to treat a tumor expressing endoplasmin. A humanendoplasmin antibody covalently linked to a radioactive isotope is ofuse to localize a tumor in radioimmunoguided surgery, such that thetumor can be surgically removed. In one embodiment, about 10 mCi of aradiolabeled human endoplasmin monoclonal antibody is administered to asubject. In other embodiments, about 15 mCi, about 20 mCi, about 50 mCi,about 75 mCi or about 100 mCi of a radiolabeled human endoplasminmonoclonal antibody is administered to a subject. In other embodiments,about 100 mCi to about 100 mCi of a radiolabled human endoplasminmonoclonal antibody is administered to a subject.

A method of detecting tumors in a subject in vivo includes theadministration of a human antibody that specifically binds endoplasmin,or antigen-binding fragment thereof, complexed to an effector molecule,such as a radioactive isotope. After a sufficient amount of time haselapsed to allow for the administered radiolabeled antibody to localizeto the tumor, the tumor is detected. In one specific, non-limitingexample, a radiolabeled immune complex is detected using a hand heldgamma detection probe. In some embodiments, the tumor is detected byMRI, CT scan or PET scan. Primary tumors, metastasized tumors, or cellsexpressing endoplasmin can be detected. For example, a human endoplasminmonoclonal antibody complexed to an effector molecule, such as aradioactive isotope, is administered to a subject prior to surgery ortreatment. In one specific embodiment, the detection step is performedprior to surgery to localize the tumor. In another embodiment, thedetection step is performed during surgery, for example to detect thelocation of the tumor prior to removing it, as in radioimmunoguidedsurgery. A human endoplasmin monoclonal antibody complexed to aneffector molecule, such as a radioactive isotope, can also beadministered to a subject following surgery or treatment, to determinethe effectiveness of the treatment, such as to ensure the completeremoval of the tumor, or to detect a recurrence of the tumor. Thus, theantibodies are of use as therapeutic agents (such as for immunotherapyagainst tumors) or for carrying out radioimmunoguided surgery.

VI. Diagnostic Methods and Kits

A method is provided herein for the detection of the expression ofendoplasmin in vitro. In one example, expression of endoplasmin isdetected in a biological sample. The sample can be any sample,including, but not limited to, tissue from biopsies, autopsies andpathology specimens. Biological samples also include sections oftissues, for example, frozen sections taken for histological purposes.Biological samples further include body fluids, such as blood, serum,plasma, sputum, spinal fluid or urine.

In several embodiments, a method is provided for detecting a malignancysuch as squamous cell carcinoma (such as head and neck squamous cellcarcinoma), melanoma, renal cancer, lung cancer, glioma, bladder cancer,ovarian cancer or pancreatic cancer. Antibodies that specifically bindendoplasmin, or antigen-binding fragments thereof, can be used to detectendoplasmin in a serum sample from a subject to detect cancer in thesubject, or confirm a diagnosis of cancer in a subject. The antibodiescan also be used to identify the original of a metastatic lesion.

The disclosure provides a method for detecting endoplasmin in abiological sample, wherein the method includes contacting a biologicalsample with a human antibody that binds endoplasmin, or anantigen-binding fragment thereof, under conditions conducive to theformation of an immune complex, and detecting the immune complex, todetect the endoplasmin in the biological sample. In one example, thedetection of endoplasmin in the sample indicates that the subject has amalignancy. In another example, detection of endoplasmin in the sampleconfirms a diagnosis of cancer in a subject. In a further example,detection of endoplasmin confirms or detects the presence of metastases.

In some embodiments, the fully human monoclonal antibody thatspecifically binds endoplasmin, or antigen-binding fragment thereof, isused for detection or diagnosis of a tumor in a subject, such asconfirming the diagnosis of a tumor in a subject. In other embodiments,the fully human monoclonal antibody that specifically binds endoplasmin,or antigen-binding fragment thereof, is used to detect the efficacy of atherapy. For example, a subject with a known malignancy that expressesendoplasmin is administered a therapeutic agent. The method can includecontacting a biological sample with a human antibody that bindsendoplasmin, or an antigen-binding fragment thereof, under conditionsconducive to the formation of an immune complex, and detecting theimmune complex, to detect the endoplasmin in the biological sample. Adecrease in the amount of endoplasmin, as compared to a control, such asa sample from the subject prior to treatment or a reference standard,indicates that the therapeutic agent is effective at treating themalignancy. In some examples, an increase in the amount of endoplasmin,as compared to the control indicates that the therapeutic agent is noteffective for treating the malignancy.

In some embodiments, the detection can be in vivo. The human monoclonalantibody that specifically binds endoplasmin, or antigen-bindingfragment thereof, can be complexed to a radioactive isotope. After asufficient amount of time has elapsed to allow for the administeredradiolabeled antibody to localize to the tumor, the tumor is detected,such as by MRI, CT scan or PET scan (see above).

In one embodiment, the human antibody that specifically bindsendoplasmin or antigen-binding fragment thereof is directly labeled witha detectable label. In another embodiment, the human antibody thatspecifically binds endoplasmin or antigen-binding fragment thereof (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the human antibody that specifically binds endoplasmin islabeled. As is well known to one of skill in the art, a second antibodyis chosen that is able to specifically bind the specific species andclass of the first antibody. For example, if the first antibody is ahuman IgG, then the secondary antibody may be an anti-human-IgG. Othermolecules that can bind to antibodies include, without limitation,Protein A and Protein G, both of which are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ³⁵S, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F, ^(99m)Tc, ¹³¹I,³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I.

In an alternative embodiment, endoplasmin can be assayed in a biologicalsample by a competition immunoassay utilizing endoplasmin standardslabeled with a detectable substance and an unlabeled human antibody thatspecifically binds endoplasmin. In this assay, the biological sample,the labeled endoplasmin standards and the human antibody thatspecifically bind endoplasmin or antigen-binding fragment thereof arecombined and the amount of labeled endoplasmin standard bound to theunlabeled antibody is determined. The amount of endoplasmin in thebiological sample is inversely proportional to the amount of labeledendoplasmin standard bound to the antibody that specifically bindsendoplasmin, or antigen-binding fragment thereof.

The immunoassays and methods disclosed herein can be used for a numberof purposes. In one embodiment, the human antibody that specificallybinds endoplasmin or antigen-binding fragment thereof may be used todetect the production of endoplasmin in cells in cell culture. Inanother embodiment, the antibody can be used to detect the amount ofendoplasmin in a biological sample. Increased expression of endoplasminis associated with several types of cancer, including, but not limitedto melanoma, breast cancer, head and neck squamous cell carcinoma, renalcancer, lung cancer, glioma, bladder cancer, ovarian cancer orpancreatic cancer. In one embodiment, a kit is provided for detectingendoplasmin in a biological sample, such as a serum sample or tissuesample. For example, to confirm a cancer diagnosis in a subject, abiopsy can be performed to obtain a tissue sample for histologicalexamination. Alternatively, a serum sample can be obtained to detect thepresence of endoplasmin protein. Kits for detecting a polypeptide willtypically comprise a human antibody that specifically binds endoplasmin,such as any of the antibodies disclosed herein. In some embodiments, anantibody fragment, such as an Fv fragment or scFv, or a Fab is includedin the kit. In a further embodiment, the antibody is labeled (forexample, with a fluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds endoplasmin. Theinstructional materials may be written, in an electronic form (such as acomputer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting endoplasmin in a biological samplegenerally includes the steps of contacting the biological sample with anantibody or antibody fragment which specifically reacts, underimmunologically reactive conditions, to an endoplasmin polypeptide. Theantibody is allowed to specifically bind under immunologically reactiveconditions to form an immune complex, and the presence of the immunecomplex (bound antibody) is detected directly or indirectly.

When the antibody is used to detect cancer or confirm diagnosis ofcancer in a subject, the information about the diagnosis can bedisplayed on a medium of expression, such as an electronic or papermedium. An electronic medium can include, for example, a computerdatabase, a display monitor, or an electronic medical record. A papermedium includes, for example, a test result or paper record as recordedby a laboratory or clinician.

In some embodiments, once a diagnosis of the tumor (such as themelanoma) is made, the subject is treated for the tumor (such as themelanoma). For example, the treatment of can include surgical excisionof a primary or metastatic lesion and/or administration of achemotherapeutic regimen for the treatment of the disease.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies can be conjugatedto other compounds including, but not limited to, enzymes, magneticbeads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs. The antibodies can also beutilized in immunoassays such as but not limited to radioimmunoassays(RIAs), enzyme linked immunosorbent assays (ELISA), orimmunohistochemical assays. The antibodies can also be used forfluorescence microscopy or fluorescence activated cell sorting (FACS). AFACS employs a plurality of color channels, low angle and obtuselight-scattering detection channels, and impedance channels, among othermore sophisticated levels of detection, to separate or sort cells (seeU.S. Pat. No. 5,061,620). Any of the human antibodies that specificallybind endoplasmin, as disclosed herein, can be used in these assays.Thus, the antibodies can be used in a conventional immunoassay,including, without limitation, an ELISA, an RIA, FACS, tissueimmunohistochemistry, Western blot or immunoprecipitation.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Convincing clinical evidence has shown that antibody-based immunotherapycan be effective in the treatment of hematological malignancies andsolid tumors. To eliminate the influence of the immunogenicity of tumorantigens on the specificity of the developed antibodies, a syntheticphage single chain variable region (scFv) library was used to isolatehuman antibodies which recognize cell-surface molecules that areup-regulated on malignant cells. Antibodies were isolated thatspecifically bind endoplasmin.

Panning of the synthetic phage scFv library with the human melanoma cellline WM1158 has resulted in the isolation of a scFv fragment, named W9,which displays high reactivity with a large panel of human cell lines.SDS-PAGE analysis of the antigen immunoprecipitated by scFv W9 from celllines identified a 94-KDa component. The determinant recognized by scFvW9 includes carbohydrates, since its expression was markedly reduced oncells incubated with tunicamycin. Mass spectrometry-based analysis ofthe band immunoprecipitated by scFv W9 from various cell linesidentified the 94-KDa component as endoplasmin, a member of the 90-KDamolecular chaperone family. This conclusion was corroborated by thereactivity of scFv W9 with the endoplasmin (Grp94) recombinant canineprotein, which displays a 98.5% homology in the amino acid sequence withhuman endoplasmin (Grp94). The determinant recognized by scFv W9 is notexpressed on normal cells. The antibody was effective in inducingapoptosis and inhibited cancer cell growth. Thus, the results disclosedherein document that antibodies that specifically bind endoplasmin, suchas scFv W9, are of use for the immunotherapy of malignant diseases.These antibodies also can be used to detect malignant disease.

Example 1 Materials and Methods

The following materials and methods were used in the below examples:

Cell Lines:

The human melanoma cell lines WM1158, MV3, COL038, SK− MEL-28, M14, andFO-1, the human breast carcinoma cell lines SUM149, MDA-MB-435s, MCF-7,T47D, the human head and neck cancer cell line PCI-13, human pancreaticcell lines Panc 2.03, Panc 3.27, Panc 10.05, the human colon cancer cellline 40-16, the human renal cancer cell line SLR21, the human prostatecancer cell line Du145, the human ovarian cancer cell line OVCAR3, thehuman glioma cancer cell line U-138, the human cervical cancer cell lineHeLa and the human B lymphoid cell line LG2 were maintained in RPMI 1640medium (Cellgro, Mediatech, Washington, D.C., USA) supplemented with 10%fetal bovine serum (FBS: BioWhittaker, Walkersville, Md., USA) and 2 mML-glutamine (BioWhittaker). The human bladder cancer cell line T24, thehuman lung cancer cell line A549, the human epidermoid cancer cell lineA431, the human glioma cancer cell line A-172 and human 293 cell linewere grown in DMEM medium (Lonza, Verviers, Belgium) supplemented with10% FBS. Cells were cultured at 37° C. in a 5% CO₂ atmosphere.

Monoclonal Antibodies, scFv Antibodies and Reagents:

The C-myc oncoprotein-specific mouse mAb 9E10 (Evan, et al., Mol CellBiol, 1985 December; 5(12):3610-6).

and the HLA-class I antigen-specific mouse mAb TP25.99 (D'Urso et al., JClin Invest. 1991 January; 87(1): 284-292) have been previouslydescribed. The anti-anti-id scFv #119 (Wang et al., 1997. Theanti-idiotypic approach to active specific immunotherapy of malignantmelanoma. In idiotypes in Medicine: Autoimmunity, Infection and Cancer.Y. Shoenfeld, R. Kennedy, and S. Ferrone, eds. Elsevier, Amsterdam, p.523) was isolated from the synthetic scFv library (#1) (Nissim et al.,1994. Embo J 13:692-698) by panning with the anti-id mAb MK2-23. MousemAb were purified from ascitic fluid by sequential ammonium sulphate andcaprylic acid precipitation (Temponi et al., 1989, Hybridoma 8:85-95).The purity and activity of mAb preparations were assessed by SDS-PAGEand by testing with the corresponding antigen in a binding assay,respectively. HRP-anti-mouse IgG Fc antibodies were purchased fromJackson ImmunoResearch (Laboratories, Inc., West Grove, Pa., USA).R-phycoerythrin (RPE)-labeled F(ab′)₂ fragments of goat anti-mouse Igantibodies were purchased from BD Pharmingen (San Diego, Calif., USA).The endoplasmin (Grp94) recombinant canine protein was purchased fromStressgen Biothecnology Corporation (Victoria, British Columbia,Canada).

Phage Display Libraries:

The semi-synthetic phage library of human single chain Fv (scFv)antibodies was constructed as described by Nissim et al. 1994, Embo J13:692-698).

Selection of Phage Display scFv Antibodies:

Phage display scFv antibodies binding to melanoma cells were isolatedfrom the phage display scFv antibody library as previously described(Noronha et al. 1998, J Immunol 161:2968-2976). Briefly phage particles(1×10³) were added to a polypropylene culture tube containing 1×10⁷WM1158 melanoma cells. Following a 90 min of incubation at R/T, unboundphages were removed by washing the cells six times with PBS. Bound phagewere eluted by adding 200 μl of 0.1M glycine-HCl (pH=2.2). Followingfour rounds of panning, the isolated clones were adsorbed against humanB-lymphoid cells, LG-2, to remove phages binding to Ags shared by humanmelanoma and lymphoid cells.

Binding Assay:

The ELISA to test the reactivity of soluble scFv W9 antibody with tumorcell lines and endoplasmin (Grp94) recombinant canine protein wasperformed as described (Noronha et al., 1998, J Immunol 161:2968-2976).Results are expressed as absorbance of optical density (O.D.) at 450 nm.

Immunoprecipitation Experiments:

WM1158 cells (3×10⁷) were washed, pelleted, and lysed in 1.5 ml of lysisbuffer (50 mmol/L Tris, 4 mmol/L EDTA, 150 mmol/L NaCl, 0.5% NP40containing 1 mmol/L phenylmethylsulfonyl fluride) containing proteaseinhibitors. Following a 30 min of incubation on ice, the cell lysate wasspun at 13,000×g for 30 min at 4° C. The supernatant was collected,precleared by incubation with PG-Sepharose (Amersham Pharmacia BiotechAB, Uppsala, Sweden), and transferred to a tube containing 15 μl ofpacked protein G Sepharose, previously armed with 15 μg of mAb 9E10 andthe periplasmic preparation of scFv W9, and 119 (negative control).Following a 2 h of incubation at 4° C., beads were washed 4 times withPBS, twice with high salt buffer (350 mmol/L NaCl, 1 mmol/L EDTA, 10mmol/L Tris, 0.1% bovine serum albumin, 1% NP40), and 2 times with lysisbuffer. Precipitated proteins were eluted in SDS sample buffer, resolvedon a reducing 12% Tris-HCl SDS-polyacrylamide gel and stained withCoomassie blue.

Tunicamycin Treatment:

COL038 cells were cultured in the presence of 0.5 μg/ml of tunicamycin(MP Biomedicals, Solon, Ohio, USA) for 72 hours at 37° C. in a 5% CO₂atmosphere. Cells incubated in medium with DMSO alone were used as acontrol.

Transfection:

293 cells were transfected with 3 μg of Grp 94 HSP90B 1 cDNA clone(Origene) using the Amaxa nucleofection technology and following themanufacturer's instructions (Amaxa, Cologne, Germany). The nucleofectorprogram Q-001 was used. After transfection, cells were immediatelysuspended in 500 μl of pre-warmed DMEM culture medium supplemented with10% FBS and plated in 6-well plates in a humidified 37° C., 5% CO₂incubator for 24 hours. Transfection efficiency was determined by flowcytometric analysis of GFP. The pCMV6-XL4 vector was used as a control.Transfection with Lipofectamine 2000 (Invitrogen, Carlsbad, Calif., USA)was performed according to the manufacturer's instructions. Thetransfection of cells with endoplasmin (Grp94) siRNA and the controlsiRNA (Fluorescein Conjugate)-A (Santa Cruz Biotechnology, Santa Cruz,Calif., USA) was carried out according to the manufacturer'sinstructions.

The following materials and methods were also used (see Examples 5 and9):

Cell Lines, Cell Lysates and Tissues.

The human melanoma cell lines M21, MV3 and SK-MEL-5, the humanpancreatic adenocarcinoma cell lines MiaPaCa-2 and PANC1, the humanglioma cell line U1338MG, the human breast carcinoma cell lines SUM149and MDA-MB-231, the human mesothelioma cell line Phi, the human coloncancer cell line RKO, the human ovarian cancer OVCAR3, the human sarcomacell line HT1080, the human multiple myeloma cell line MM.8, the human Blymphoid cell line RAJI, and the mouse myeloma cell line NSO weremaintained in RPMI 1640 medium supplemented with 2 mM L-glutamine(Cellgro) and 10% fetal bovine serum (FBS) (PAA Laboratories Inc). Cellswere cultured at 37° C. in a 5% CO2 atmosphere. Cell lysates wereprepared as described (Desai et al. Cancer Res 1998; 58(11):2417-25).

Animals.

C.B-17 SCID mice (8-10 weeks old) were obtained from Taconic Farms, Inc.

Monoclonal and Polyclonal Antibodies, scFv Antibodies and Reagents.

The

HLA class I antigen-specific mouse mAb TP25.99 (Desai et al., J Immunol2000; 165(6):3275-83), the calnexin-specific mAb TO-5 (used as a loadingcontrol) were developed and characterized as described (Ogino et al.,Tissue Antigens 62:385-393, 2003). Purified human immunoglobulins werepurchased from Sigma-Aldrich. Antibodies specific for FAK andphosphorylated FAK (Tyr397) and for ERK1/2 and phosphorylated 44/42ERK1/2, AKT and phosphorylated 473 AKT, MET phosphorylated MET, PKC,β-catenin, Ras, B-Raf, C-Raf, cleaved Caspase-3, cleaved Caspase-7 SHh,GLI1 and β-actin were purchased from BD Bioscience and from Cellsignaling technology. The rat-anti-endoplasmin (Grp94) antibody waspurchased from StressGen. Mouse mAb were purified from ascitic fluid bysequential ammonium sulphate and caprylic acid precipitation (Temponi etal., Hybridoma 1989; 8(1):85-95.). The purity and activity of mAbpreparations were assessed by SDS-PAGE and by testing with thecorresponding antigen in a binding assay, respectively.

HRP-anti-mouse, -rabbit, and rat antibodies and RPE-labeled F(ab′)₂fragments of goat anti-human IgG Fcγ antibody were purchased fromJackson ImmunoResearch Laboratories Inc. RPE-labeled F(ab′)₂ fragmentsof goat anti-mouse Ig antibodies were purchased from BD Pharmingen.

Construction of Fully Human mAb W9.

The gene encoding scFv W9 variable light (VL) and heavy (VH) regionswere amplified by PCR and cloned into the pFUSE2-CLIg-hk andpFUSE-CHIg-hG1, respectively (InvivoGen), utilizing the DNA LigationKit, MIGHTY MIX® (TAKARA Bio USA) according to the manufacturer'sinstructions.

Fully Human mAb W9 Expression and Purification.

Expression plasmids pFUSE2-CLIg-hk and pFUSE-CHIg-hG1 wereco-transfected into the mouse myeloma cell line NSO usingelectroporation (GENE PULSER® II Electroporation System Bio-Rad)according to the manufacturer's instructions. The transfected cells wereselected in RPMI 1640 medium supplemented with 10% FCS, Zeocin (50μg/mL), and Blasticidin S (10 μg/mL). Cells resistant to Zeocin andBlasticidin S were then single-cell-subcloned by limiting dilution. Thespent supernatants of subcloned cells were screened by ELISA for theexpression of human Fc and (Fab′)₂ and for the reactivity with thecorresponding antigens. The fully human mAb W9 was purified from eitherspent culture supernatant or mouse ascites, using HITRAP® protein G HPcolumn (GE healthcare) according to the manufacturer's instructions. Thepurity and activity of purified mAb W9 was determined by SDS-PAGE andantigen binding assays, respectively.

Endoplasmin (Grp94) deglycosaylation.

Grp94⁺ MIAPaCa-2 cells (5×10⁵) were incubated with or without 2 μlPNGase F, 2 μl O-Glycosidase and 2 μl α-2(3,6,8.9)-Neuraminidase(Enzymatic Protein Degylcosylation Kit, Sigma) in 50 μl RPMI1640 mediumfor 24 hours at 37° C. The treated cells were then stained with mAb W9and analyzed by flow cytometry (Cyan, Beckman Coulter).

Flow Cytometry Analysis.

Cells (2×10⁵) were incubated for 30 min at 4° C. with 2 μg/ml of mAb W9(diluted in a total volume of 100 μl of 2% BSA-PBS). Cells were thenwashed twice with 0.5% BSA-PBS and incubated for 30 min at 4° C. with anoptimal amount of RPE-labeled F(ab′)₂ fragments of goat anti-human IgGFcγ antibody (Jackson ImmunoResearch, Inc). Following three washes,cells were fixed in 2% formaldehyde and analyzed with a CYAN™ ADP LX 9Color flow cytometer (Dako). mAb TP25.99 and human immunoglobulins wereused as a control. For cancer initiating cell binding assay cells werepreviously stained with ALDEFLUOR® (Stem Cell Technologies) followingthe manufacturer's instructions.

Immunohistochemistry.

Frozen sections of surgically removed human pancreatic adenocarcinomalesion and normal pancreas tissues were fixed by 4% formaldehyde/PBS for20 minutes at room temperature. IHC staining of TMA slides withscFv-FcC21 was performed as described (Wang et al., Curr. Mol. Med.2010). Pictures of stained tissue microarrayslides were taken usingOLYMPUS®BX51 microscope (OLYMPUS UK Ltd) at a magnification ×200 forreview.

Cell Proliferation and MTT Assays.

Cells were seeded at a density of 1×10⁴ per well in 96-well plates wereincubated with mAb W9 (5 μg/ml) in medium supplemented with 1% FCS for 3days. The viable cell numbers at different time points were measured byadding 10 μl per well of tetrazolium componentmethylthiazolyldiphenyl-tetrazolium bromide (Sigma-Aldrich, Inc. StLouis, Mo.) and the mixture was incubated for approximately 3˜4 hours at37° C. Metabolically active, viable cells converted MTT into a coloredformazan product that was measured in a spectrophotometric microplatereader (MTX Lab System, Inc, Vienna, Va.) at 540 nm. The results wereexpressed as percent inhibition of living cells, using the number ofliving cells incubated with PBS only as a 100% reference.

Apoptosis.

Flow cytometry analysis of apoptotic and necrotic MV3 and MIAPaca-2cells following a 6 hr incubation with mAb W9 (50 μg/mL) was performedby Annexin V-FTIC and propidium iodide (PI) staining kit (BDPharMingen), as per the manufacturer's specifications.

Western Blot.

Proteins in cell lysates were separated by 8% sodium dodecylsulfate—polyacrylamide gel electrophoresis (SDS-PAGE) and transferredonto 0.45-μm (pore size) PVDF membranes (Millipore). After blocking with5% nonfat dry milk plus 2% BSA overnight at 4° C., membranes weresequentially incubated with the appropriate concentration of primaryantibodies for overnight at 4° C. and HRP-labeled respective secondaryantibodies for 45 min at room temperature. Bands were visualized withthe enhanced chemiluminescence system (GE Life Science), and banddensity was read with the FOTO/Analyst® Investigator Eclipse system(Fotodyne Incorporate). The calnexin-specific mAb TO-5 and β-actinspecific mAb were used as loading control.

Immunoprecipitation. MV3 cells (3×10⁷) were washed, pelleted, and lysedin 1.5 ml of lysis buffer (50 mmol/L Tris, 4 mmol/L EDTA, 150 mmol/LNaCl, 0.5% NP40 containing 1 mmol/L phenylmethylsulfonyl fluride)containing protease inhibitors. Following a 30 minute incubation on ice,the cell lysate was spun at 13,000×g for 30 min at 4° C. The supernatantwas collected, precleared by incubation with PG-Sepharose (AmershamPharmacia Biotech AB, Uppsala, Sweden), and transferred to a tubecontaining 10 μl of packed protein G Sepharose, previously armed with 10μg of mAb mAb W9. Following 2 hours of incubation at 4° C., beads werewashed 4 times with PBS, twice with high salt buffer (350 mmol/L NaCl, 1mmol/L EDTA, 10 mmol/L Tris, 0.1% bovine serum albumin, 1% NP40), and 2times with lysis buffer. Precipitated proteins were eluted in SDS samplebuffer, resolved on a reducing 12% Tris-HCl SDS-polyacrylamide gel andtransferred onto 0.45-1 μm (pore size) PVDF membranes (Millipore). Theblotting was performed as previously described.

ADCC:

MV3 cells were labeled with 50 μCi of ⁵¹Cr (Perkin Elmer) andresuspended at a density of 0.4×10⁶ cells/ml. ⁵¹Cr labeled cells weremixed with mAb W9 (50, 10, and μg/ml 50 μl/well) in a 96-well tissueculture-U-bottom assay plate (BD. Falcon). Human immunoglobulins wereused as a control. Following a 30 minute-incubation at 4° C., PBMC (40:1effector to target (E:T)) were added and incubated for 4 hours at 37° C.in a CO₂ incubator. ⁵¹Cr release was determined by counting the cellfree supernatant using Packard TOPCOUNT™ Microplate ScintillationCounter (Conroe). The experiment was performed twice in triplicate.

CDC.

Target cells MV3 were labeled with 50 μCi of ⁵¹Cr and resuspended at adensity of 1×10⁶ cells/ml. MV3 cells were incubated with mAb W9 (50, 10,and μg/ml 50 μl/well) in presence of human serum complement (Quidel)diluted four times in RPMI 1640, 10 mM HEPES, 0.1% BSA. Humanimmunoglobulins were used as a control. Following a 2-hour incubation at37° C. in a CO₂ incubator, ⁵¹Cr release was determined by counting thecell free supernatant using Packard TOPCOUNT™ Microplate ScintillationCounter. The experiment was performed twice in triplicate.

Treatment of Mice Bearing Established Human Melanoma Cell-Derived LungMetastasis.

Eight-week-old, female SCID mice were injected intravenously (i.v.) withthe human melanoma MV3 cells (1.4×10⁸ cells/mouse). Fifteen daysfollowing the i.v. injection of cells, mice were randomly divided intotwo groups of 13 mice each. One group of mice was injected i.v. with mAbW9 (100 μg/per mouse) every 48 hours for a total of 3 injections. Theother group of mice was injected i.v. with human immunoglobulins,utilizing the same schedule. On day 25 mice were sacrificed, lungs werecollected and subjected to FFPE. H&E stained lung tissue sections wereexamined microscopically for metastasis.

Statistical Analysis.

The statistical significance of the difference between the resultsobtained in the tested groups was analyzed using the Student's t-test.Survival statistics was analyzed using MEDCALC® software (Mariakerke,Belgium).

Example 2 Isolation of Melanoma Cell-Binding scFv Fragments

The synthetic phage scFv library (#1) was subjected to four rounds ofpanning with WM1158 cells. Isolated phages were absorbed with culturedhuman LG-2 B lymphoid cells to remove the phages binding to the Agsshared by human cells. Soluble scFvs were produced from 80 clones andtested for reactivity with melanoma cells in a cell ELISA. Among theclones tested, the scFv named W9, strongly reacted with the WM1158 cellline. The reactivity was specific since no reactivity with LG-2 cellswas detected. The scFv #119, which recognizes an irrelevant antigen, wasused as a negative control (FIG. 1).

Example 3 Reactivity of scFv W9

When tested by ELISA with a panel of human cell lines, the soluble scFvW9 reacted with melanoma (MV3, COL038, SK-MEL-28, M14, FO-1), basalbreast cancer (SUM149, MDA-MB-435s), head and neck (PCI-13), pancreatic(PANC 2.03, PANC 3.27, PANC 10.05), bladder (T24), lung (A549),epidermoid (A431), cervical (HeLa), renal (SLR21), ovarian (OVCAR3), andglioma (U-138, A-172) cancer cell lines. The scFv fragment did not reactwith the luminal breast cancer cell lines (MCF-7, T47D), the coloncancer cell line 40-16, the prostate cancer cell line Du145, and the Blymphoid cell line LG-2 (FIG. 2).

The results from an immunohistochemical analysis of scFv W9immunoreactivity with normal tissues is presented below. Results arerelated to biopsies of at least two patients tested (⁺Positive).

TISSUES* W9 Skin sweat gland only: + Kidney negative Lung negative Livernegative Colon-rectum negative Pancreas negative Stomach negativeThyroid negative Brain Cortex negative Testis negative Parotid acinarepithelium only: + Breast negative Prostate negative Spleen negative

Immunohistochemical staining with scFv W9 showed that endoplasmin(Grp94) is expressed by sweat gland and acinar epithelium only while itis not expressed by a variety of normal tissues.

Immunohistochemical staining with mAb W9 showed that the endoplasmin(Grp94) epitope has a restricted distribution in normal tissue and isexpressed in pancreatic adenocarcinoma lesion (FIG. 11). Theimmunohistochemical analysis has shown that the epitope recognized bymAb W9 has a restricted distribution in normal tissues. Moreover, mAb W9stained only the surgically removed human pancreatic adenocarcinomalesion while it didn't stain the normal pancreas tissue from the samepatient. Furthermore MDA-MB-231 and MV3 cell line-derived xenograft,respectively, were strongly stained by mAb W9 while no staining wasdetected in the MCF-7 (FIG. 12).

Example 4 Immunochemical Analysis of the Specificity of scFv W9 andIdentification of Grp94 by Tandem Mass Spectrometry

The immunoprecipitation of a total cell lysate obtained from WM1158awith scFv W9 resulted in the identification of a unique band atapproximately 100 KDa (FIG. 3). scFv #119 was used as a negativecontrol. The same results were obtained utilizing lysates from MV3(melanoma), SUM149 (basal breast cancer), T24 (bladder cancer), andSLR21 (renal cancer) cell lines. The 100 KDa specific bandsimmunoprecipitated by scFv W9 from the different cell lysates wereexcised and in-gel digested with trypsin. The analysis of resultingtryptic peptides by liquid chromatography-tandem mass spectrometryidentified two tryptic peptides (ELISNASDALDK (SEQ ID NO: 7) andGVVDSDDLPLNVSR (SEQ ID NO:8)) that are derived uniquely from endoplasmin(Grp94) (FIG. 4).

The epitope recognized by mAb W9 is critically dependent on sialic acidresidue(s), since its reactivity with tumor cells was abrogatedfollowing incubation with neuraminidase, but was not affected by otherglycosidases (FIG. 9). For these experiments, human pancreaticadenocarcinoma MIAPaCa-2 (5×10⁵) were incubated with or without 2 μl ofα-2(3,6,8.9)-Neuraminidase in 50 μl RPM I1640 medium for 24 hours at 37°C. in a 5% CO₂ incubator. The treated cells were then stained with mAbW9 and analyzed by flow cytometry. Cells treated with mAb TP25.99 wereused as a control.

Human pancreatic adenocarcinoma MIAPaCa-2 cells were incubated withALDEFLUOR® to detect ALDH activity (TEST), and stained with mAb W9.Cells incubated with ALDEFLUOR®+DAEB inhibitor and stained with mAb W9were used as a reference (CONTROL). Human Ig (HIg) were used as acontrol. The percentage of cancer initiating cells, identified asALDH^(bright) cells, is indicated. Flow analysis showed that the epitoperecognized by mAb W9 is expressed by cancer initiating cells since thesame antibody bound to the identified ALDH bright cell population (FIG.10).

Example 5 Effect of Tunicamycin on the Expression of the DeterminantRecognized by scFv W9 and Further Characterization of the Epitope

Glycosylation plays a role in the expression of the epitope recognizedby scFv W9 on endoplasmin (Grp94), since this scFv fragment did notreact with COL038 cells treated with tunicamycin, an inhibitor ofN-glycosylation of glycoproteins (FIG. 4). No inhibition was observedwith DMSO alone. Furthermore no inhibition was observed for TP25.99 mAb(control) under the same conditions. These data suggest thatcarbohydrates are essential for the recognition of endoplasmin (Grp94)by scFv W9.

The epitope recognized by mAb W9 is critically dependent on sialic acidresidue(s), since its reactivity with tumor cells was abrogatedfollowing incubation with neuraminidase, but was not affected by otherglycosidases (FIG. 9).

Example 6 Reactivity of scFv W9 with Endoplasmin (Grp94) CanineRecombinant Protein

scFv W9 specifically reacted with the endoplasmint (Grp94) caninerecombinant protein (RC-Grp94) in a dose dependent fashion (FIG. 5A),since no binding was detected with BSA (negative control, FIG. 5B).Furthermore RC-Grp94 specifically affected the binding of scFv W9 toCOL038 cells in a dose dependent fashion. The inhibition was dosedependent and specific, since β2-microglobuline (negative control)displayed no inhibitory effect (FIG. 6).

Example 7 Effect of Electroporation on Binding of scFv W9

Electroporated 293 cells were transiently transfected with endoplasmin(Grp94) full length cDNA or the vector alone (pCMV-XL4, negativecontrol). The transfection efficiency (94%) was assessed by GFPexpression. Cells harvested 24 hours after transfection were incubatedwith scFv W9 and mAb 9E10, followed by incubation with FITC-goatanti-mouse IgG antibodies. Cells were analyzed by flow cytometry.Untransfected cells were used as a control. A strong increase in thebinding of scFv W9 was observed in cells treated with endoplamsin(Grp94) (FIG. 7A) and with the plasmid alone (FIG. 7B). These datasuggests that the binding of scFv was increased by electroporation andthat the heat shock regulates the expression of the antigen recognizedby scFv W9.

Example 8 Effect of shRNA Targeted Against Endoplasmin (Grp94) onBinding of scFv W9

FO-1 cells were transduced with either shRNA to achieve knockdown ofendoplasmin (Grp94), or ABCB5 shRNA as control. After 72 hours fromtransduction cells were harvested, incubated with scFv W9 and mAb 9E10,followed by incubation with FITC-goat anti-mouse IgG antibodies. Cellswere analyzed by flow cytometry. Grp94 shRNA (FIG. 8A) significantlyinhibited the binding of scFv W9 compared with the control shRNA (FIG.8B).

Example 9 Effect of mAb W9 on Cancer Cells

The effect of mAb W9 on cancer cells was assessed. Specifically, MTTanalysis has shown that mAb W9 significantly inhibited the growth of allendoplamsin positive (Grp94+) tumor cell lines tested. However, noeffect was observed in a Grp94⁻ Raji cell line. The anti-proliferativeeffect was specific, since no growth inhibition was observed with humanimmunoglobulins used as a control (FIG. 13). Thus, mAb W9 inhibitedcancer cell proliferation.

Flow analysis has shown that mAb W9 induced apoptosis in human melanomaMV3 and pancreatic cancer MIAPaCa-2 cells (48.90% and 53.56% ofapoptosis respectively). Furthermore, Western blot analyses showed asignificant increase of the expression of cleaved Caspase-3 and cleavedPARP in cells treated with mAb W9 (FIGS. 14, 15 and 16). Thus, mAb W9induced apoptosis in cancer cells. However, mAb W9 did not mediatecell-nor complement-dependent lysis of target cells (FIGS. 17 and 18).

In addition, mAb W9 inhibited proliferation of human pancreaticadenocarcinoma MIAPaCa-2 cancer initiating cells. Flow analysis hasshown that in cells treated with mAb W9, the percentage of cancerinitiating cells, defined as ALDH^(bright) cells, was reduced by 50%when compared with human immunoglobulins (negative control) (FIG. 19).mAb W9 treated cells had a decreased level of Ras, C-Raf, PCKα,β-catenin, and Bcl-2. Moreover, mAb W9 treatment inhibited theactivation of Akt, Erk, Mek, Fak, and Met (FIG. 20-22). In a pull-downassay using mAb W9, endoplsmin (Grp94) was co-immuno-precipitated withMet, Ras, and C-Raf (FIG. 23).

H&E stained tissue sections were analyzed for the cumulative area of themetastatic nodules present in 5 randomly selected, 200× fields/sectionusing an OLYMPUS® BX51 microscope (OLYMPUS® UK Ltd.). SPOT IMAGINGSOFTWARE® Advanced (Diagnostic Instruments, Inc.) was used to measureand calculate the mean tumor area for each group, and these values forMV3 tumor are provided in the bar graphs ±SD. mAbW9 treated mice had astatistically significant (about 50%) reduction in the area ofmetastases compared to that treated with the isotype control antibody(FIG. 24).

Endoplasmin (Grp94) expression was enhanced by chemotherapeutic agents.Flow analysis has shown that treatment with 5-fluorouracil (FU),Cisplatin, and Paclitaxel increased the surface expression ofEndoplasmin (Grp94). The effect was specific (FIG. 25). MTT resultsshowed that mAb W9 in combination with 5-FU and cyclopamine,respectively, is more effective than each agent alone in inhibitingpancreatic adenocarcinoma cell growth (FIG. 26). Flow analysis alsoshowed that inhibition of cancer initiating cell growth by mAb W9 wasenhanced by cyclopamine and 5-FU. Approximately 90% growth inhibition ofALDH^(bright) cells was detected after incubation with mAb W9,cyclopamine and 5-FU. In contrast, the inhibition was only 50% in thecultures incubated with mAb W9 or cyclopamine individually, 20% in theculture incubated with 5-FU and 70% in the cultures incubated with mAbW9 in combination with cyclopamine or 5-FU (FIG. 27). Flow analysis alsoshowed that the induction of apoptosis by mAb was enhanced bycyclopamine and 5-FU. Apoptosis was induced in 70% of cells by mAb W9 incombination with cyclopamine and 5-FU. However, apoptosis was induced inless than 35% of the cells treated with 5-FU, cyclopamine, or acombination of 5-FU and cyclopamine (FIG. 28).

Flow analysis also showed that inhibition of cancer initiating cellgrowth by mAb W9 was enhanced by radiation and cyclopamine.Approximately 90% growth inhibition of ALDH^(bright) cells was detectedafter treatment with mAb W9, in combination with radiation andcyclopamine. In contrast only the inhibition was approximately 50% inthe cultures treated with mAb W9 or cyclopamine individually, and lessthan 30% in the cultures only treated with radiation (FIG. 29). Flowanalysis showed that the induction of apoptosis by mAb was enhanced byradiation and cyclopamine. Apoptosis was induced in 73.87% of cells bymAb W9 in combination with radiation and cyclopamine, but in less than25% by mAb W9 alone (FIG. 30).

The effect of mAb W9 treatment on Ras and C-Raf protein levels was alsoexamined. Ras and C-Raf levels were enhanced by cyclopamine and 5-FU. Asynergistic effect was found in the inhibition of the activation of Mek,Erk, and Akt (FIG. 31). The effect of mAb W9 treatment in inhibiting Rasand GLI1 protein levels as well as the activation of Erk, and Akt wasenhanced by radiation and cyclopamine (FIG. 32).

Example 10 Endoplasmin-Specific Monoclonal Antibodies for the Treatmentof Cancer

This example describes the use of endoplasmin-specific human monoclonalantibodies for the treatment of cancers that exhibit overexpression ofendoplasmin (referred to herein as a “endoplasmin-positive” cancer),including, but not limited to melanoma, breast cancer, head and necksquamous cell carcinoma, renal cancer, lung cancer, glioma, bladdercancer, ovarian cancer or pancreatic cancer. Patients diagnosed with anendoplasmin-positive cancer can be treated according to standardprocedures in the art. Generally, treatment options include surgery,radiation therapy, chemotherapy, immunotherapy or interferon therapy.

In this example, patients diagnosed with an endoplasmin-positivemelanoma are administered an immunoconjugate comprising anendoplasmin-specific human monoclonal antibody linked to Pseudomonasexotoxin (PE). Preparation of PE immunoconjugates has been described(see, for example, U.S. Pat. No. 7,081,518 and U.S. Pre-GrantPublication No. 2005/0214304, which are herein incorporated byreference). In some patients, the immunoconjugate is administered byintravenous bolus injection every other day for a total of three to sixdoses. In other patients, the immunoconjugate is administered bycontinuous intravenous infusion over the course of ten days. The dose ofimmunoconjugate administered to a patient varies depending on the weightand gender of the patient, and mode and time course of administration.Following treatment, patients are evaluated for cancer progression(including tumor growth and metastasis) and other clinical signs ofillness. Patients can be treated with the immunoconjugate alone, or incombination with one or more standard cancer treatments. For example, apatient that has undergone surgery to remove the melanoma cansubsequently be treated with the immunoconjugate.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An isolated human monoclonal antibody or an antigen-binding fragmentthereof comprising a heavy chain and a light chain, wherein the heavychain comprises amino acids 26-33 of SEQ ID NO: 1 (CDR1), amino acids51-58 of SEQ ID NO: 1 (CDR2), amino acids 97-103 of SEQ ID NO: 1 (CDR3),or a combination of two or three thereof, and wherein the antibodyspecifically binds human endoplasmin.
 2. The isolated human monoclonalantibody, or antigen-binding fragment of claim 1, wherein the lightchain of the antibody comprises amino acids 27-32 of SEQ ID NO: 2(CDR1), amino acids 50-52 of SEQ ID NO: 2 (CDR2), amino acids 89-97 ofSEQ ID NO: 2 (CDR3), or a combination of two or three thereof.
 3. Theisolated human monoclonal antibody or antigen-binding fragment of claim1, wherein the heavy chain variable domain of the antibody comprises SEQID NO:
 1. 4. The isolated human monoclonal antibody or antigen-bindingfragment of claim 1, wherein the light chain variable domain of theantibody comprises SEQ ID NO:
 2. 5. The isolated human monoclonalantibody, or antigen-binding fragment of claim 1, wherein theantigen-binding fragment is a Fab fragment, a Fab′ fragment, a F(ab)′₂fragment, a single chain Fv protein (scFv), or a disulfide stabilized Fvprotein (dsFv).
 6. The isolated human monoclonal antibody orantigen-binding fragment of claim 1, wherein the antibody is an IgG. 7.The isolated human monoclonal antibody or antigen-binding fragment ofclaim 1, wherein the antibody is labeled.
 8. The isolated humanmonoclonal antibody or antigen-binding fragment of claim 1, wherein (a)the antibody specifically binds glycosylated endoplasm, and (b) theisolated human monoclonal antibody or antigen-binding fragment competesfor binding to the glycosylated endoplasm with a second antibodycomprising a heavy chain and a light chain, wherein the heavy chain ofthe second antibody comprises the amino acid sequence set forth as SEQID NO: 1 and the light chain of the second antibody comprises the aminoacid sequence set forth as SEQ ID NO:
 2. 9. A composition comprising theisolated antibody, or antigen-binding fragment of claim 1, and apharmaceutically acceptable carrier.
 10. An isolated chimeric moleculecomprising the human monoclonal antibody or antigen-binding fragment ofclaim 1 linked to an effector molecule.
 11. The isolated chimericmolecule of claim 10, wherein the effector molecule is a cytokine or achemokine.
 12. The isolated chimeric molecule of claim 11, wherein thecytokine is interferon-alpha.
 13. The isolated chimeric molecule ofclaim 12, wherein the human monoclonal antibody or antigen bindingfragment comprises a heavy chain and a light chain, wherein the heavychain comprises amino acids 26-33 of SEQ ID NO: 1 (CDR1), amino acids51-58 of SEQ ID NO: 1 (CDR2) and amino acids 97-103 of SEQ ID NO: 1(CDR3); and wherein the light chain comprises amino acids 27-32 of SEQID NO: 2 (CDR1), amino acids 50-52 of SEQ ID NO: 2 (CDR2), and aminoacids 89-97 of SEQ ID NO: 2 (CDR3).
 14. The isolated chimeric moleculeof claim 13, wherein interferon alpha is linked to the human monoclonalantibody or antigen binding fragment with a peptide linker.
 15. Acomposition comprising the isolated chimeric molecule of claim 11 and apharmaceutically acceptable carrier.
 16. A method of treating a subjectdiagnosed with cancer that expresses endoplasmin, comprisingadministering to the subject a therapeutically effective amount of theantibody of claim 1, thereby treating the cancer that expressesendoplasmin in the subject.
 17. The method of claim 16, wherein thecancer is a melanoma, breast cancer, head and neck squamous cellcarcinoma, renal cancer, lung cancer, glioma, ovarian cancer, bladdercancer or pancreatic adenocarcinoma.
 18. A method of detecting cancer orconfirming the diagnosis of cancer in a subject, comprising: contactinga sample from the subject with the isolated human monoclonal antibody,or antigen-binding fragment of claim 1; and detecting binding of theisolated human monoclonal antibody, or antigen-binding fragment to thesample, wherein an increase in binding of the isolated human monoclonalantibody, or antigen-binding fragment to the sample as compared tobinding of the isolated human monoclonal antibody, or antigen-bindingthereof, to a control sample detects cancer in the subject or confirmsthe diagnosis of cancer in the subject.
 19. The method of claim 18,wherein the cancer is melanoma, breast cancer, head and neck squamouscell carcinoma, renal cancer, lung cancer, glioma, bladder cancer,ovarian cancer or pancreatic cancer.
 20. An isolated or recombinantnucleic acid molecule encoding the human monoclonal antibody orantigen-binding fragment of claim
 1. 21. An expression vector comprisingthe isolated or recombinant nucleic acid molecule of claim
 20. 22. Anisolated host cell transformed with the nucleic acid molecule of claim21.